STUDIES OF CYTOCHROME C OXIDASES FROM RHODOBACTER ...

STUDIES OF CYTOCHROME C OXIDASES FROM RHODOBACTER SPHAEROIDES

AND VIBRIO CHOLERAE

BY

HUAZHI HAN

DISSERTATION

Submitted in partial fulfillment of the requirements

for the degree of Doctor of Philosophy in Biophysics and Computational Biology

in the Graduate College of the

University of Illinois at Urbana-Champaign, 2010

Urbana, Illinois

Doctoral Committee:

Professor Robert B. Gennis, Chair

Professor Antony R. Crofts

Professor James H. Morrissey

Professor Yi Lu

ii

ABSTRACT

Cytochrome c oxidase is the terminal enzyme to accept electrons from cytochrome c in

the respiratory chain of mitochondria, bacteria and archaea. It couples the reducing oxygen to

water and pumping the protons across the membrane. The second largest group, C-type, is

also known as cbb3 cytochrome c oxidase which is almost exclusively found in the

Proteobacteria. All the current studies show that cytochrome cbb3 oxidases apply quite

distinct mechanism for receiving electrons and pumping protons. The work in this thesis

clearly demonstrates that B- and C-type oxidases have less energy coupling efficiency and

share a similar pumping mechanism other than A-type family members. Subunits CcoO and

CcoP in cbb3–type oxidase comprise of one and two heme c respectively. The binding motif

for heme c is characterized to be the sequence of CXXCH. Site-directed mutagenesis studies

of the various combined mutants of the motif show that the cysteine and histidine residues are

critical for the incorporation of heme c into cbb3 enzymes from Rhodobacter sphaeroides and

Vibrio cholerae. Results also show that every heme c is involved in the electron

translocations in a consequential way and is important for the activity and stability of the

enzyme complex. Sequence alignment and structural modeling have shown that cbb3–type

oxidase contains only one proton channel (K-channel analogue) which consists of its own

conserved residues. The putative K-channel related residues were tested by site-directed

mutagenesis and all shown to be important for catalytic activity. ICP-OES analysis in this

study demonstrates that only two calcium ions exist as the redox inactive metal groups. One

identified Ca2+

connects subunit CcoN and CcoO and bridges heme b and heme b3 through

iii

the propionate groups. Site-directed mutagenesis studies of the binding residues E180

(subunit CcoN) and S105 (subunit CcoO, R. sphaeroides numbering) indicate that they are

essential for the activity of the enzyme and recruitment of both calcium ions and CuB.

Moreover, mutagenesis study of another conserved nearby residue E183 suggests that this

acidic residue is also required for the correct incorporation of calcium and copper ions and

necessary for the fully functional enzyme.

iv

TABLE OF CONTENTS

LIST OF ABBREVIATIONS ............................................................................................................ v

CHAPTER 1: INVESTIGATION OF PROTON PUMPING EFFICIENCIES OF CYTOCHROME

C OXIDASES THROUGH WHOLE CELL PUMPING ASSAY .................................................... 1

1.1 Abstract .............................................................................................................................. 1

1.2 Introduction ........................................................................................................................ 1

1.3 Materials and methods ....................................................................................................... 4

1.4 Results ................................................................................................................................ 6

1.5 Discussion .......................................................................................................................... 9

1.6 Figures and tables ............................................................................................................. 12

1.7 References ........................................................................................................................ 18

CHAPTER 2: MUTAGENESIS STUDY OF HEME C BINDING SITES IN CBB3 OXIDASES

FROM RHODOBACTER SPHAEROIDES AND VIBRIO CHOLERAE ........................................ 23

2.1 Abstract ............................................................................................................................ 23

2.2 Introduction ...................................................................................................................... 23

2.3 Materials and methods ..................................................................................................... 26

2.4 Results .............................................................................................................................. 29

2.5 Discussion ........................................................................................................................ 32


2.7 References ........................................................................................................................ 40

CHAPTER 3: COMPARATIVE GENOMICS AND SITE-DIRECTED MUTAGENESIS

SUPPORT THE EXISTENCE OF ONLY ONE INPUT CHANNEL FOR PROTONS IN THE

C-FAMILY (CBB3 OXIDASE) OF HEME-COPPER OXYGEN REDUCTASES * ..................... 45

3.1 Abstract ............................................................................................................................ 45

3.2 Introduction ...................................................................................................................... 46


3.4 Results .............................................................................................................................. 52

3.5 Discussion ........................................................................................................................ 58

3.6 Conclusions ...................................................................................................................... 65


3.8 References ........................................................................................................................ 74

CHAPTER 4: ROLE OF CALCIUM BINDING SITES IN CBB3–TYPE CYTOCHROME C

OXIDASE ....................................................................................................................................... 86

4.1 Abstract ............................................................................................................................ 86

4.2 Introduction ...................................................................................................................... 86


4.4 Results .............................................................................................................................. 92

4.5 Discussion ........................................................................................................................ 95


4.7 References ...................................................................................................................... 104

v

LIST OF ABBREVIATIONS

Å angstrom (10-10

meters)

AMP ampicillin

ATP adenosine triphosphate

bp base pair

CCCP carbonyl cyanide m-chlorophenylhydrazine

CcO cytochrome c oxidase

CO carbon monoxide

COV cytochrome oxidase vesicle

Da Dalton

DEAE diethylaminoethyl

DDM dodecyl--D-maltoside

DNA deoxyribonucleic acid

E. coli Escherichia coli

EDTA ethylenediaminetetraacetic acid

EGTA ethylene glycol tetraacetic acid

HEPES 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid

Kan kanamycin

KPi potassium phosphate

LB Luria-Bertani broth

Mg milligram

mL milliliter

µg microgram

vi

µL microliter

MW molecular weight

NADH reduced nicotinamide adenine dinucleotide

Ni-NTA nickel nitrilotriacetic acid

nm nanometer

PMSF phenyl methyl sulfonyl fluoride

Sp spectinomycin

St streptomycin

Tet tetracycline

TMPD N,N,N',N'-tetramethyl-p-phenylenediamine

UV ultraviolet

WT wild type

1

CHAPTER 1: INVESTIGATION OF PROTON PUMPING EFFICIENCIES OF CYTOCHROME C OXIDASES THROUGH WHOLE CELL PUMPING ASSAY

1.1 Abstract

Cytochrome c oxidases are terminal protein complexes in the aerobic

respiratory chain and widely found in Eukarya, Bacteria and Archaea. Three mainly

classified oxygen reductases, A-, B- and C- type, couple the reduction of oxygen to

the translocation of protons with different efficiencies. Here we report the modified

whole cell pumping measurement using N,N,N',N'-tetramethyl-p-phenylenediamine

(TMPD) as the sole exogenous substrate and demonstrate that stoichiometry of H+/e

-

coupling efficiency of A-type family is 1 and the number for B- and C-type families is

0.5. All of the strains tested here uniquely express only single functional oxygen

reductase which fulfills the requirement of proton pumping assay. It is concluded that

B- and C-type oxidases have less energy coupling efficiency and share a similar

pumping mechanism other than A-type family members.

1.2 Introduction

The respiratory chain of mammalian mitochondria or bacteria is mainly

composed of several multiple-polypeptide complexes of which three (I, III and IV)

function as oxidation-reduction driven proton pumps to build up a transmembrane

electrochemical potential [1, 2]. Once the substrates like NADH or succinate are

reduced, the electrons pass through the dehydrogenases to quinol pool and then

reduce the complex III. The reduced bc1 complex transfers the electrons to

cytochrome c and subsequently reduces cytochrome c oxidase in which the oxygen is

reduced to water. The series of proton pumps convert the electron transfer energy to a

membrane potential gradient which provides the free energy for ATP synthesis. The

proton pumping process could also be uncoupled by proton back-flow mechanism to

2

dissipate the energy.

Cytochrome c oxidase belongs to the heme-copper oxidase superfamily and is

the terminal enzyme to accept electrons from cytochrome C in the respiratory chain of

mitochondria and aerobic bacteria and archaea. The enzyme catalyzes the reduction of

oxygen to water and oxidation of cytochrome c. It also couples the reducing oxygen

to water and pumping the protons across the membrane [3-5]. While the electrons are

transferred to oxygen, the protons are pumped from the inside of the membrane to the

outside of the membrane by the enzyme. The overall catalyzed reaction is

O2 + 4e- + 4H

+ + nH

+IN = 2 H2O + nH

+OUT (1)

where the subscripts IN and OUT denote the cytoplasm and periplasm, respectively,

for bacterial and archaeal enzymes. Cytochrome c oxidase is integral membrane

protein complex and is composed of several subunits (varied from 2 to 13) of which

subunit I shares a high similarity and contains the redox center. The main subunit I

commonly has one low-spin heme, one heme-copper binuclear center and a

tyrosine-histidine cross-link. The rest of the subunits behave as the electron donors or

stabilizers of the complex.

Cytochrome c oxidase has been classified into three major families based on

heme contents, structural, phylogenic and genomic analyses: A-, B- , C- type families

of oxygen reductases [6, 7]. They all accept electrons from the cytochrome c but

contain different heme groups and various numbers of subunits. Since cytochrome aa3

(A-type) is the most widely characterized cytochrome c oxidase, it has always been

used as the model system to study redox reaction and pumping mechanism.

Historically A-type family is defined because of the heme a groups in subunit I which

receives the electrons from the CuA center in subunit II [8-11]. Likely wise B-type

family oxidase (ba3-like oxidase) also gets electrons from the subunit II which is

3

homologous to subunits II of the A-type family. But it consists of three subunits of

which subunit IIa has uniquely different feature as it is analogous to the second

transmembrane helix in subunit II from the A-type family[12, 13]. C-type family

oxidase (cbb3 oxidase) is the most distant member of the cytochrome c oxidase. This

type of enzyme incorporates cofactor heme c into subunits II and III and utilizes heme

b as the redox center [14, 15]. The high affinity for oxygen of cbb3 oxidase is

consistent with its functions in the Proteobacteria which can survive in low oxygen

conditions [16].

All of cytochrome c oxidases have been shown to pump protons even though

with different proton translocation channels. The A-type family oxidase conserves the

two different proton pumping pathways, called the D-channel and the K-channel. The

D-channel transports two of the chemical protons plus all four of the pumped protons

to the active site, whereas the K-channel conducts two chemical protons coupled with

two electron transfers [17-20]. Sequence alignment and mutagenesis studies have

shown that there is no analogue D-channel in B and C-type family oxidases [14, 21].

Both of these two families utilize only one proton input channel, analogue to

K-channel in A-type family, to deliver the chemical protons to form H2O and all the

pumped protons.

It has been experimentally well demonstrated that the A-type family

cytochrome c oxidase pumps one proton per electron during the oxygen reduction

process [22-26]. Therefore, the number of n in equation 1 should be 4. However, the

B-type family (ba3-type oxidase) appears to pump only half as many protons per

catalytic cycle [27]. And the number of n in equation 1 becomes 2. There has never

been a fixed amount of protons pumped during each cycle for C-type cytochrome c

oxidase. Variable H+/e

- stoichiometry numbers from 0.1 to 1 have been reported in the

4

past either by whole cell suspension method or purified cytochrome cbb3 oxidase

reconstituted into phospolipid vesicles [28-32]. Here we describe the modified intact

whole cell pumping method which was used to analyze the pumping efficiency of the

different cytochrome c oxidases. This is the first quantitative stoichiometry report for

all the three different kinds of cytochrome c oxidases measured in parallel conditions.

Our data consistently show that A-type family cytochrome c oxidase pumps 4 protons

per catalytic cycle, whereas B- and C- type families pump 2 protons per dioxygen

molecule consumed.

1.3 Materials and methods

Bacteria strains and growth conditions. All the different Rhodobacter capsulatus,

Rhodobacter sphaeroides, Thermus thermophilus and Helicobater pylori strains used

in this work are described in Table 1. R. capsulatus strains were grown on

magnesium–calcium, peptone, yeast extract (MPYE) enriched medium at 32 °C and

supplemented with kanamycin or spectinomycin (10 or 20 µg per ml, respectively)

[33]. R. sphaeroides mutant strain was grown semiaerobically at 30 °C in Sistron

media with 2 µg per ml tetracycline and 50 µg each of streptomycin and

spectinomycin per ml [28]. T. thermophilus strains were grown at 60 °C in Thermus

LB with 50 µg kanamycin/ml or 10 µg bleomycin/ml [34]. H. pylori was grown at

37°C under a 5% oxygen-5% carbon dioxide-90% nitrogen atmosphere in bisulfite-

and sulfite-free brucella broth supplemented with 5% fetal bovine calf serum and 5 µg

of vancomycin/ml.

Purification of Recombinant Protein. The expression and purification of the R.

sphaeroides cbb3 oxygen reductase was performed as previously described [35].

Whole cell Oxidase Activity Measurements. The steady-state oxidase activity was

measured by a YSI model 53 oxygen monitor. The reaction mixture buffer used for

5

oxidase activity measurements was 1 mM Hepes, 150 mM KCl and 100 mM KSCN at

pH 7.4. 0.5 mM N,N,N',N'-tetramethyl-p-phenylenediamine (TMPD) was used as

exogenous reductant to initiate the oxygen reduction reaction. 100 µM KCN was used

as the oxidase inhibitor to stop the reaction. And 10 mM ascorbate was added to test if

the reaction could be resumed. The reactions were performed in the water-jacketed

chamber with stir bar at 25 °C. The reaction buffer was mixed with 50 mg/ml cells at

the beginning to start as the base line. Oxygen consumption was monitored upon

adding the exogenous reductants.

Preparation of proteoliposomes. Purified cytochrome cbb3 oxidase from R.

sphaeroides was reconstituted into phospholipid vesicles as previously described [29].

The RCR (respiratory control ratio) estimation was then performed to check the

integrity of the vesicles [36, 37].

Proton pumping analysis. The reconstituted phospholipid vesicles with purified cbb3

oxidase (2µM from Rhodobacter sphaeroides were incubated anaerobically together

with valinomycin(5µM), ascorbate (500µM) and horse heart cytochrome c (30µM) in

60mM KCl buffer solution. The proteoliosomes were pulsed with 10µl air saturated

pure water at 25 °C. Calibration was done by the same amount of 1mM HCl. Upon

adding 10 µM CCCP, the same pulse procedure was repeated again to show the

negative control.

Whole cell pumping experiments were done by mixing the starved intact cells

with buffer containing 150mM KCl, 100mM KSCN and 0.5mM HEPES (pH7.4).

TMPD was used as the electron donor (1mM final concentration). The same pulse

procedure was followed. Up to 100µM KCN or 10µM Myxothiazol or 10 µM CCCP

was added to the solution to testify if the proton translocation was due to cytochrome

c oxidase.

6

1.4 Results

Proton pumping assay by proteoliposomes

Cytochrome cbb3 oxidase from R. sphaeroides was overexpressed in the aa3 and

cbb3 deletion strain with expression plasmid pUI2803NHIS as previously described

[38]. After passing through the nickel affinity column cbb3 oxidase was further

purified by FPLC system using ion-exchange column. The SDS-PAGE gel shows the

purity of the enzyme with subunits CcoN, CcoO and CcoP positioned at 40-, 32- and

25- kDa respectively (Fig.1). Heme-staining gel for heme c further confirms the

integrity of the protein complex. Following the established reconstitution procedures

for aa3 type cytochrome c oxidase we introduced the purified enzyme into the

liposomes. The respiratory control ratio (RCR) was shown to be ~3 which indicated

the integrity of the vesicle (Fig.2). Air saturated pure water was pulsed into the mixed

proteoliosomes and reduced substrates solution to testify the pumping efficiency of

cbb3 oxidase. But only alkalization phenomena was observed which suggests the

protons were pumped inside into the vesicles (Fig.2). This could be due to the wrong

natural orientation of the enzyme when they were incorporated into the liposome. In

the mean time purified aa3 oxidase from R. sphaeroides was used as the control

system to ensure the valid procedure and the proton pumping ability of aa3 was shown

to be ~0.5 H+/e

-(not shown). In such a case, we are urged to perform the alternative

whole cell pumping analysis.

Selection of strains

In order to compare the pumping efficiencies of different classes of cytochrome

c oxidases, over 1000 bacteria strains were screened to select the suitable clean

system for the analysis. Thermus thermophilus turned out be ideal bacteria as it

7

contains only two different oxidases caa3 and ba3 which represent the A- and B- type

family respectively. Knocking out either one oxidase made the perfect strain carrying

only one oxidase. This step is critical as any kind of background noise including

quinol oxidases might mess up the results easily. Rhodobacter capsulatus is another

good candidate as it comprises one quinol oxidase and one cbb3 oxidase. As a

consequence the quinol oxidase was knocked out to get the derivative strain which

contains only C- type cytochrome c oxidase. Naturally there is only one cbb3 type

oxidase in Helicobater pylori which is perfect for the proton pumping assay. So the

wildtype strain was used for the test. Rhodobacter sphaeroides has three quinol

oxidases, one aa3 and one cbb3 type oxidase. After the aa3 and cbb3 oxidase were

knocked out the other three quinol oxidases still played around. The overexpression

plasmid pUI2803NHIS was cloned into the strain to overproduce cbb3 oxidase. Under

anaerobic conditions most majority of the oxidases are cbb3 type due to the surviving

pressure. With these unique strains whole cell pumping experiments were carried out

to give the comparative analysis under parallel conditions.

Whole cell turn over activity test

All the different deletion strains were tested with the bc1 complex inhibitor

Myxothiazol and cytochrome c oxidase inhibitor KCN for the sensitivity assay. For T.

thermophilus and H. polyri strains after twice washing with proton pumping buffers

there were no detectable turn over without exogenous substrate for reduction. This

indicates that the fast metabolisms of these strains could consume all the endogenous

substrates within pretty short period. Afterwards TMPD alone was used to feed the

suspended cells and oxygen concentration level started to fall. In the mean time the

visible TMPD color slowly changed to blue which indicated the oxidation of TMPD.

This suggests that TMPD alone could be the exogenous electron donor for the oxygen

8

reduction reaction by oxidase. It is worth to mention that at higher capacity of buffer

(50mM phosphate) the turn over was much faster than in the proton pumping buffer

(0.5mM Hepes). This is due to the proton gradient built up by oxygen reduction

reactions. As all the cytochrome c oxidases have been shown to be sensitive to

cyanide fewer than 100 µM KCN was used to inhibit the reaction. As expected all the

oxygen reduction processes were fully stopped for all the strains upon the adding of

potassium cyanide. In contrast it’s hard to control the starvation time for R. capsulatus

and R. sphaeroides strains. Longer exposure to the buffer in 30 °C caused the suicide

of the cells or the degradation of enzymes. Within shorter incubation time endogenous

substrate could not be used up. The least starved cells were used to test the activity

with leftover endogenous substrates. The initial reactions were monitored by the

oxygen electrode. About 10µM Myxothiazol was used to inhibit the bc1 complex,

therefore the oxygen reduction was stopped. This confirms that the endogenous

substrates pass the electrons through bc1 complex to the cytochrome c oxidases. As

expected artificial electron donor TMPD could donate the electrons to cytochrome c

oxidases and resume the oxygen reduction with the presence of bc1 complex inhibitor.

After adding the potassium cyanide the cytochrome c oxidases were killed and the

oxygen reduction reactions were stopped. R. capsulatus GK32 strain only has one

quinol oxidase and it was used as the control system. The oxygen uptake reactions by

quinol oxidases could not be inhibited by potassium cyanide up to 1 mM range. As

shown in Figure 3A ascorbate resumed the oxygen uptake reaction somehow with the

presence of cyanide in R. capsulatus, R. sphaeroides and T. thermophilus strains.

Therefore it was excluded in the all the whole cell pumping experiments.

Whole cell proton pumping assay

It is very important to keep the system background as clean as possible while

9

performing the intact whole cell pumping assay. Not only all of the strains used are

unique with only one kind of oxidase, but also TMPD alone was used as the only

reductant. Ascorbate was excluded in the reaction because it caused false signals

which were cyanide insensitive and it released protons during the reaction (Fig. 3).

Myxothiazol was added the mixture solution to ensure no electrons could be

transferred through bc1 complex to cytochrome c oxidases. After anaerobiosis by

argon within short period different amounts of oxygen saturated water (5-25µl ) were

pulsed before TMPD was fully oxidized which could be indicated by the color change.

And the proton translocation was determined by the pH alteration which was

monitored by the sensitive pH electrode. Same amounts of 1mM HCl were added to

compensate the protons consumed. And the pH change caused by HCl was used as the

calibration system to calculate the proton pumping efficiency. In T. thermophilus caa3

oxidase was shown to pump protons around 1.1 H+/e

- using ba3 oxidase knocked out

strain and ba3 oxidase was shown to pump around 0.5 H+/e

- using caa3 oxidase

knocked out strain (Table 2). These results are quite consistent with previously

reported. Whereas in R. capsulatus, R. sphaeroides and H. polyri strains all the cbb3

type oxidases were shown to translocate protons 0.4-0.5 H+/e

- (Table 2) All of the

cytochrome c oxidases were shown to be cyanide sensitive fewer than 100 µM range.

These results strongly suggest that B- and C-type oxidases function as proton pumps

in a similar way other than A-type oxidases.

1.5 Discussion

Almost all of the purified known A-type cytochrome c oxidases have been

shown to pump protons with the ratio of H+/e

- to one unit. In cell suspensions the

stoichiometry reports from different groups have always been consistent. However,

10

complementary experiments with purified cytochrome c oxidases reconstituted into

phospholipids vesicles demonstrating that H+/e

- stoichiometry approaches to 1 have

only been shown by several groups. Due to the natural orientation of the enzyme

embedded into liposomes many groups reported the maximum efficiency was less

than 0.7. This does not change the overall conclusion that A-type oxidases pump one

proton per electron. Our whole cell pumping assay for caa3 oxidase from T.

thermophilus indicates that this principle also applies to caa3 oxidase and intact whole

cell measurement is more reliable regarding to quantitative purpose.

For B-type oxidase, proton pumping has been demonstrated with H+/e

-

stoichiometry of 0.5 for ba3 oxidase from T. thermophilus using reconstituted vesicles.

Similar phenomena like aa3 oxidase have been observed in other groups. The

numbers obtained in our group are always around 0.2-0.3 by the proteolipids vesicles

method using pH potentiometric or stopped-flow spectrophotometric techniques. Here

we reproducibly demonstrate that the H+/e

- ratio for ba3 type oxidase is 0.5 using

whole cell pumping measurement.

So far only two groups have successfully shown purified cbb3 -type oxidases

from Bradyrhizobium japonicum and Helicobater pylori could pump protons using

the methods of reconstituted phospholipids vesicles. But the precise values of H+/e

-

ratio were not reported for Helicobater pylori and the stoichiometry numbers varied

from 0.2-0.4 for B. japonicum. We failed to detect any pumping signals through

purified cbb3 oxidase from R. sphaeroides even with the control experiment for aa3

oxidase. Similar results happened in Mårten Wikström’s group through oral

conversation. Whole cell pumping assay for C-type oxidases has only been performed

in Paracoccus denitrificans and R. sphaeroides by three groups. When succinate was

used as the exogenous substrate proton translocation H+/e

- ratio could reach to 1,

11

however when succinate was replaced by ascorbate and TMPD the value of this ratio

fell substantially. For cbb3 oxidase from R. sphaeroides the value varied from 0.6-1.1,

but the strain used contains quinol oxidase which could contribute the pumping effect.

Since succinate donates electrons to cytochrome c oxidases through quinol pool and

bc1 complex which also releases protons, TMPD should be a more suitable substrate

for pumping analysis as it could provide electrons directly to cbb3 oxidases. Normally

ascorbate is coupled to keep TMPD reduced, but it releases protons while being

oxidized. Plus ascorbate always gives false pumping signals which are not cyanide

sensitive which is also true in activity assay. So in our experiment TMPD alone was

used as the direct electron donor to cytochrome c oxidases for the oxygen reduction.

Our results provide the most direct evidence that cbb3 -type oxidases pump protons

with 0.4-0.5 H+/e

-.

Therefore we fill the n in equation 1 as 4 for A-type oxidases and as 2 for B-

and C-type oxidases which strongly suggest that B- and C-type share more similarity

in terms of functionality and structure. Homology modeling and mutagenesis studies

have shown that B- and C-type oxidases only have one proton input channel whereas

A-type family contains two proton input channels. Our study is consistent with the

physiological studies that B- and C-type oxidases are less efficient at transducing

energy than A-type oxidase.

12

1.6 Figures and tables

Table 1.1. Bacteria strains used in this study

Strains Relevant characteristics Ref./source

R. capsulatus

R. capsulatus Wildtype [33]

GK32 cbb3-

KZ1 Qox-

a

T. thermophilus HB8

T. thermophilus HB8 Wildtype [34]

YC_1001 ba3-

KG100 caa3-

b

R. sphaeroides 2.4.1

R. sphaeroides 2.4.1 Wildtype [28]

JR1000 aa3- , cbb3

- with plasmid

pUI2803NHIS

c

H. polyri

H. pylori 700392 Wild-type d

a Provided by Prof. Dr. Fevzi Daldal (unpublished)

b Provided by Krithika Ganesan in Prof. Robert Gennis’ lab (unpublished)

c Provided by Prof. Jung Hyeob (unpublished)

d Provided by Dr. Vijay Gupta in Prof. Steven Blanke’s lab

13

Table 1.2. Whole cell proton pumping assay of different types of oxidases from

various strains.

Cytochrome c

oxidase

Source strain TMPD O2 (H+/e

-)

caa3 T. thermophilus HB8 1.14 + 0.12

ba3 T. thermophilus HB8 0.52 + 0.07

cbb3

H. pylori 700392 0.44 + 0.05

R. capsulatus 0.41 + 0.04

R. sphaeroides 2.4.1 0.53 + 0.06

H+/e

- ratios are averages + standard deviation (n=10). Exogenous substrate was

TMPD. Anaerobic suspensions were pulsed with air-saturated H2O. Cells were

cultivated as text described.

14

Figure 1.1

Fig. 1.1. (A) SDS-PAGE pattern of the purified R. sphaeroides cbb3–type oxidase

(10 μg). The concentration of polyacrylamide in the gel was 12.5%. The gel was

stained with Coomassie Brilliant Blue R-250. (B) The same PAGE gel was

stained with TMBZ to detect Heme c. (C) Reduced minus oxidized difference

spectrum of R. sphaeroides cbb3 oxidase.

15

Figure 1.2

coupled

uncoupled

CCCP+Val

H2O

HCl

Time(s)

A

B

Fig. 1.2. (A) Respiratory control ratio measurement of proteoliposomes reconstituted

form R. sphaeroides cbb3 oxidase. Oxygen uptake was measured in the presence of 30

µM horse heart cytochrome c with coupled state and uncoupled state which contains

CCCP and valinomycin. (B) Change in pH upon O2 pulse by proteoliposomes

reconstituted form R. sphaeroides cbb3 oxidase. 10 µL of air-saturated water

containing about 0.25 mM dioxygen, equilibrated at 25oC, was added to the reaction

chamber to initiate the reaction. Afterwards, 10 µL of anaerobic 1 mM HCl was added

as a calibration.

16

Figure 1.3

AB

C D

Fig. 1.3. (A) Oxygen uptake by R. capsulatus KZ1 strain. 0.5 mM TMPD was added

to the cell suspension to initiate the reaction. 100 µM potassium cyanide was added to

stop the reaction. 10mM ascorbate was added to test if the oxygen consumption could

be resumed. (B) Change in pH upon O2 pulse by suspended R. capsulatus KZ1 strain.

3 consecutive 10 µL of air-saturated water was injected into the solution followed by

10 µL of anaerobic 1 mM HCl injection 2 times. (C) Same pH measurement like (B)

after adding 10 µM CCCP. (D) pH change monitored upon adding 100 µM KCN.

17

Figure 1.4

A B

C D E

0 5 10 15 20 25

0.00

0.01

0.02

0.03

0.04

0.05

0.06

20l

10l

15l

5l

HCl

ExpFit

H2O

p

H

H+(nm)

T. th. caa3

0 5 10 15 20 25

0.00

0.01

0.02

0.03

0.04

0.05

0.06

T. th. ba3

p

H

H+(nm)

20l

10l

25l

15l

5l

HCl

ExpFit

H2O

0 5 10 15 20 25

0.00

0.01

0.02

0.03

0.04

0.05 H. pylo. cbb3

p

H

H+(nm)

20l

10l

15l

25l

5l

HCl

ExpFit

H2O

0 5 10 15 20

0.000

0.005

0.010

0.015

0.020

0.025

0.030

R. sph. cbb3

5l

10l

20l

p

H

H+(nm)

HCl

ExpFit

H2O

0 5 10 15 20 25

0.00

0.01

0.02

0.03

0.04

0.05

HCl

ExpFit

H2O

pH

H+(nm)

R. cap. cbb3

5l

10l

15l

20l25l

Fig. 1.4. Change in pH upon different amounts of O2 pulse by various bacteria strains

carrying different types of oxidases. pH changes caused by oxygen were calibrated by

same amounts of HCl. (A) T. thermophilus HB8 YC_1001 strain. (B) T. thermophilus

HB8 KG100 strain. (C) H. pylori 700392 wildtype strain. (D) R. capsulatus KZ1

strain. (E) R. sphaeroides 2.4.1 JR1000 strain.

18

1.7 References

1. Belevich, I. and Verkhovsky, M. I. (2008) Molecular mechanism of proton

translocation by cytochrome c oxidase, Antioxid. Redox Signal. 10, 1–30.

2. Hosler J. P., Ferguson-Miller S., and Mills D. A. (2006) Energy transduction:

Proton transfer through the respiratory complexes, Annu Rev Biochem 75, 165–187.

3. Wikstrom, M., and Verkhovsky, M. I. (2006) Towards the Mechanism of Proton

Pumping by the Haem-Copper Oxidases, Biochim. Biophys. Acta 1757, 1047-1051.

4. Michel, H. (1998) The Mechanism of Proton Pumping by Cytochrome c Oxidase,

Proc. Natl. Acad. Sci. U.S.A. 95, 12819-12824.

5. Garcia-Horsman, J. A., Barquera, B., Rumbley, J., Ma, J., and Gennis, R. B. (1994)

The Superfamily of Heme-Copper Respiratory Oxidases, J. Bacteriol. 176,

5587-5600.

6. Hemp J., Gennis R. B. (2008) Diversity of the heme-copper superfamily in Archae:

Insights from genomics and structural modeling, Results Problems Cell

Differentiation 1–31.

7. Pereira, M. M., Santana, M., and Teixeira, M. (2001) A Novel Scenario for the

Evoluation of Haem-copper Oxygen Reductases, Biochim. Biophys. Acta 1505,

185-208.

8. Wikström, M. (1977) Proton Pump Coupled to Cytochrome c Oxidase in

Mitochondria, Nature 266, 271-273.

9. Pereira, M. M., Verkhovskaya, M. L., Teixeira, M., and Verkhovsky, M. I. (2000)

The caa3 Terminal Oxidase of Rhodothermus marinus Lacking the Key Glutamate of

the D-Channel Is a Proton Pump, Biochemistry 39, 6336-6340.

19

10. Yoshikawa, S., Shinzawa-Itoh, K., and Tsukihara, T. (1998) Crystal Structure of

Bovine Heart Cytochrome c Oxidase at 2.8 Å Resolution, J. Bioenerg. Biomembr. 30,

7-14.

11. Ostermeier, C., Harrenga, A., Ermler, U., and Michel, H. (1997) Structure at 2.7 Å

Resolution of the Paracoccus denitrificans Two-Subunit Cytochrome c Oxidase

Complexed with an Antibody Fv Fragment, Proc. Natl. Acad. Sci. U.S.A. 94,

10547-10553.

12. Kannt, A., Soulimane, T., Buse, G., Becker, A., Bamberg, E., and Michel, H.

(1998) Electrical Current Generation and Proton Pumping Catalyzed by the ba3-type

Cytochrome c Oxidase from Thermus thermophilus, FEBS 434, 17-22.

13. Soulimane, T., Buse, G., Bourenkov, G. P., Bartunik, H. D., Huber, R., and Than,

M. E. (2000) Structure and mechanism of the aberrant ba(3)-cytochrome c oxidase

from thermus thermophilus, Embo J. 19, 1766-76.

14. Hemp, J., Han, H., Roh, J. H., Kaplan, S., Martinez, T. J., and Gennis, R. B. (2007)

Comparative genomics and site-directed mutagenesis support the existence of only

one input channel for protons in the C-family (cbb3 Oxidase) of heme-copper oxygen

reductases, Biochemistry 46, 9963-9972.

15. Pitcher, R. S., and Watmough, N. J. (2004) The bacterial cytochrome cbb3

oxidases, Biochim. Biophys. Acta 1655, 388-399.

16. Preisig, O., Zufferey, R., Thöny-Meyer, L., Appleby, C. A., and Hennecke, H.

(1996) A high affinity cbb3-type cytochrome oxidase terminates the symbiosis

specific respiratory chain of Bradyrhizobium japonicum, J. Bacteriol. 178,

1532–1538.

20

17. Namslauer, A., Pawate, A., Gennis, R. B., and Brzezinski, P. (2003)

Redox-coupled proton translocation in biological systems: proton shuttling in

cytochrome c oxidase, Proc. Natl. Acad. Sci. U.S.A. 100, 15543-15547.

18. Gennis, R. B. (2004) Coupled proton and electron transfer reactions in

cytochrome oxidase, Front. Biosci. 9, 581-591.

19. Gennis, R. B. (1998) Multiple Proton-conducting Pathways in Cytochrome

Oxidase and a Proposed Role for the Active-Site Tyrosine, Biochim. Biophys. Acta

1365, 241-248.

20. Pfitzner, U., Hoffmeier, K., Harrenga, A., Kannt, A., Michel, H., Bamberg, E.,

Richter, O.-M. H., and Ludwig, B. (2000) Tracing the D-Pathway in Reconstituted

Site-Directed Mutants of Cytochrome c Oxidase from Paracoccus denitrificans,

Biochemistry 39, 6756-6762.

21. Chang, H. Y., Hemp, J., Chen, Y., Fee, J. A., and Gennis, R. B. (2009) The

cytochrome ba3 oxygen reductase from Thermus thermophilus uses a single input

channel for proton delivery to the active site and for proton pumping, Proc. Natl.

Acad. Sci. U.S.A. 106, 16169-16173.

22. Antonini, G., Malatesta, F., Sarti, P., and Brunori, M. (1993) Proton pumping by

cytochrome oxidase as studied by time-resolved stop-flow spectrophotometry, Proc.

Natl. Acad. Sci. U. S. A. 90, 5949–5953.

23. Krab, K., and Wikstrom, M. (1978) Proton-translocating cytochrome c oxidase in

artificial phospholipid vesicles, Biochim. Biophys. Acta 504, 200–214.

24. Fetter, J. R., Qian, J., Shapleigh, J., Thomas, J. W., Garcia-Horsman, A., Schmidt,

E., Hosler, J., Babcock, G. T., Gennis R. B., and Ferguson-Miller, S. (1995) Possible

proton relay pathways in cytochrome c oxidase, Proc. Natl. Acad. Sci. U. S. A. 92,

1604–1608.

http://www.ncbi.nlm.nih.gov/pubmed?term=%22Chang%20HY%22%5BAuthor%5D&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVAbstract

http://www.ncbi.nlm.nih.gov/pubmed?term=%22Hemp%20J%22%5BAuthor%5D&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVAbstract

http://www.ncbi.nlm.nih.gov/pubmed?term=%22Chen%20Y%22%5BAuthor%5D&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVAbstract

http://www.ncbi.nlm.nih.gov/pubmed?term=%22Fee%20JA%22%5BAuthor%5D&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVAbstract

http://www.ncbi.nlm.nih.gov/pubmed?term=%22Gennis%20RB%22%5BAuthor%5D&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVAbstract

21

25. Pereira, M. M., Verkhovskaya, M. L., Teixeira, M., and Verkhovsky, M. I. (2000)

The caa(3) terminal oxidase of Rhodothermus marinus lacking the key glutamate of

the D-channel is a proton pump, Biochemistry 39, 6336–6340.

26. Honnami, K., and Oshima, T. (1984) Purification and characterization of

cytochrome c oxidase from Thermus thermophilus HB8, Biochemistry 23, 454–460.


(1998) Electrical current generation and proton pumping catalyzed by the ba3-type

cytochrome c oxidase from Thermus thermophilus, FEBS Lett. 434, 17–22.

28. Toledo-Cuevas, M., Barquera, B., Gennis, R. B., Wikström, M., and

García-Horsman, J. A. (1998) The cbb3-type cytochrome c oxidase from Rhodobacter

sphaeroides, a proton-pumping heme-copper oxidase, Biochim. Biophys. Acta 1365,

421–434.

29. Arslan, E., Kannt, A., Thöny-Meyer, L., and Hennecke, H. (2000) The

symbiotically essential cbb(3)-type oxidase of Bradyrhizobium japonicum is a proton

pump, FEBS Lett. 470, 7–10.

30. de Gier, J. W., Schepper, M., Reijnders, W. N., van Dyck, S. J., Slotboom, D. J.,

Warne, A., Saraste, M., Krab, K., Finel, M., Stouthamer, A. H., van Spanning, R. J.,

van der Oost, J. (1996) Structural and functional analysis of aa3-type and cbb3-type

cytochrome c oxidases of Paracoccus denitrificans reveals significant differences in

proton pump design, Mol. Microbiol. 20, 1247–1260.

31. Raitio, M., and Wikström, M. (1994) An alternative cytochrome oxidase of

Paracoccus denitrificans functions as a proton pump, Biochim. Biophys. Acta 1186,

100–106.

22

32. Tsukita, S., Koyanagi, S., Nagata, K., Koizuka, H., Akashi, H., Shimoyama, T.,

Tamura, T., and Sone, N. (1999) Characterization of a cb-type cytochrome c oxidase

from Helicobacter pylori, J. Biochem. 125, 194–201.

33. Koch, H. G., Hwang, O., and Daldal, F. (1998) Isolation and characterization of

Rhodobacter capsulatus mutants affected in cytochrome cbb3 oxidase activity, J

Bacteriol 180, 969–78.

34. Chen, Y., Hunsicker-Wang, L. M., Pacoma, R. L., Luna, E., and Fee, J. A. (2005)

A homologous expression system for obtaining engineered cytochrome ba3 from

Thermus thermophilus HB8, Protein Expression Purif. 40, 299–318

35. Oh, J.-I., and Kaplan, S. (2002) Oxygen adaptation: the role of the CcoQ subunit

of the cbb3 cytochrome c oxidase of Rhodobacter sphaeroides 2.4.1., J. Biol. Chem.

277, 16220-16228.

36. Casey, R. P. (1986) Measurement of the H+ pumping activity of reconstituted

cytochrome oxidase, Methods Enzymol. 126, 13–21.

37. Müller, M., Thelen, M., O'Shea, P., and Azzi, A. (1986) Functional reconstitution

of proton-pumping cytochrome-c oxidase in phospholipid vesicles, Methods Enzymol.

126, 78–87.

38. Oh, J. I. (2006) Effect of Mutations of Five Conserved Histidine Residues in the

Catalytic Subunit of the cbb3 Cytochrome c Oxidase on Its Function, J. Microbiol. 44,

284-292.

23

CHAPTER 2: MUTAGENESIS STUDY OF HEME C BINDING SITES IN CBB3 OXIDASES FROM RHODOBACTER SPHAEROIDES AND VIBRIO CHOLERAE

2.1 Abstract

In cbb3-type oxidase subunit CcoO contains one heme C whereas subunit

CcoP has two covalently attached. CXXCH is the diagnostic motif of those hemes

which play an important part in the electron delivery and biogenesis of the protein

complex. Site-directed mutagenesis studies of the various combined mutants of the

motif showed that the cysteine and histidine residues are critical for the incorporation

of heme C into cbb3 enzymes from Rhodobacter sphaeroides and Vibrio cholerae.

The inactive sub-complex CcoN-CcoO formed by mutant H126A from CcoP gave the

hint that electrons might first be accepted from the subunit CcoP then to CcoO and

finally transferred to catalytical site in CcoN. All of the heme C are involved in the

electron translocations in a consequential way. The conserved histidine residues

coordinating the cofactors heme B and copper in CcoN were mutated to alanine to

verify the importance of these metal cofactors. The oxidase could not assemble

without heme B or copper. All of these results suggest that heme B, heme C and

copper are all essential for the stability and functionality of cbb3–type oxidases.

2.2 Introduction

Heme-copper oxidases (HCOs) are the terminal members in the respiratory

chain of eukaryotes and most aerobic bacteria and archaea. These enzyme complexes

couple the reducing oxygen to water and pumping the protons across the periplasmic

membrane which provides the free energy for ATP synthesis [1]. The superfamily of

HCOs could be classified into two main sub-groups depending on the substrates

where they receive electrons from [2, 3]. One subgroup belongs to quinol oxidases

24

which accept electrons directly from quinols in the cytoplasmic membrane and are

represented by cytochrome bo3 from Escherichia coli. The other subgroup contains

various cytochrome c oxidases (CcO) which receive electrons from the soluble

cytochrome c and are widely spread in both eukaryotic and prokaryotic organisms.

Cytochrome c oxidases could be further divided into three distinct families (A, B and

C) based on the sequence alignments, structural analysis, proton channels and proton

pumping efficiencies [4, 5]. Currently the most commonly found and mostly

characterized CcO is the A-type family which is about 71% of the oxygen reductases.

A-type family is represented by aa3 cytochrome c oxidase which has always been

used as the model system to study redox reaction and pumping mechanism. B-type

oxidase is normally referred as the ba3 cytochrome c oxidase. The second largest

group is the C-type (24%) which is represented by cbb3 cytochrome c oxidase and is

almost exclusively found in the Proteobacteria [2]. All the current studies show that

cytochrome cbb3 oxidases apply quite distinct mechanism for receiving electrons and

pumping protons. Their high affinity to oxygen explains the important roles that cbb3

oxidases play in low-oxygen growth conditions [6].

Two different proton input channels have been identified for aa3 oxidase based

on high resolution X-ray structures and mutagenesis studies [7-9]. D-channel is

responsible for transferring the chemical and pumped protons and K-channel is

capable of translocating the chemical protons to the binuclear center for the redox

reaction. However, in B- and C-type oxidases only K-channel has been found to fulfill

the role of moving both substrate and pumped protons [10, 11]. And more

interestingly both B- and C-type oxidases are less efficient in coupling the proton

pumping to oxygen reduction [12, 13].

Subunit I is the only common subunit for all of the HCOs and defines the

25

superfamily by the contained heme contents. Six fully conserved histidines coordinate

the low-spin haem and bimetallic catalytic center which is composed of a high-spin

haem and a copper ion [7]. The electrons are transferred from the low-spin haem to

the catalytic center for redox reaction. X-ray crystallography and mass spectrometry

have shown in the aa3-type oxidase a fully conserved tyrosine residue in the active

site forms a covalent bond to one of the three histidine ligands of CuB [14-17]. And

they are in the same helix VI and only several residues apart. However, the recently

identified tyrosine residue from the cross-linked tyrosine-histidine in the active site of

the cbb3–type oxidase is located in another helix VII [18-20].

The genes encoding cbb3 oxidases were first named fixNOQP because their

expression is required to support symbiotic N2 fixation [21]. The expression of the

four subunits ccoNOQP is positively regulated by ccoGHIS which is also a four

subunits complex [22-24]. Subunit CcoN, corresponding to subunit I of other types of

oxidases, contains the active site and has 12 helices. The binuclear center consists of a

high-spin heme b3 which couples to CuB. And another low-spin heme b in the subunit

CcoN acts as an electron transferor to the active site for oxygen reduction [25-27].

Unlike the aa3-type oxidases, subunits CcoO and CcoP corresponding to subunit II

and III contain one and two c-type hemes respectively which could serve to transfer

electrons from soluble cytochrome c donors to the catalytic subunit CcoN [27, 28].

In-frame deletion of either of these two heme c containing subunits resulted the loss

of enzyme activity and a defect in enzyme assembly [29]. The function of the last

small subunit CcoQ is still unknown although some evidence suggests it’s related to

the enzyme’s stability [27, 30, 31]. Elimination of this subunit was shown to have no

effect of the catalytic properties or the assembly of the enzyme complex.

Sequence alignments have shown that conserved heme c binding motifs

26

CXXCH exist in CcoO and CcoP as an indication of the covalent binding sites of

c-type hemes. Even though both of these two subunits could help to transfer the

electrons to CcoN, it is still not clear how the electrons are delivered sequentially.

There is no clear evidence to show if CcoO and CcoP could transfer electrons

independently or from one to another. To address this question mutagenesis study of

the heme c binding sites could provide valuable information about the functionality of

these subunits. Previous works have shown that heme c is necessary for the synthesis

of CcoO and CcoP and thus the maturation of the cbb3 protein complex [32-34]. Here

in this work we analyze the importance of heme c binding by mutating the conserved

motif sites in R. sphaeroides and V. cholerae. These studies indicate that the

biogenesis of cbb3 oxidases from R. sphaeroides and V. cholerae requires heme c

incorporation and the functional enzyme complexes need the presence of subunits

CcoO and CcoP with intact heme c binding environment. In the mean time

mutagenesis studies of the heme b and copper binding residues in CcoN showed that

these metal cofactors were prerequisites for the assembly of the enzyme.


Mutagenesis of the cbb3-Type Oxidase from R. sphaeroides. Site-directed mutagenesis

was carried out using the expression plasmid pUI2803NHIS [30]. pUI2803NHIS

contains six histidine codons before the stop codon of subunit ccoN. Point mutations

of subunit ccoO and ccoP were made by recombinant PCR and then subcloned into

pUI2803NHIS. The resulting mutant expression plasmids were transferred from E.

coli S-17-1 to R. sphaeroides 2.4.1 ccoNOQP deletion strain by conjugation.

Sequence verification of the mutagenesis products was performed at the Molecular

Genetics Core Facility, Department of Microbiology and Molecular Genetics, The

27

University of Texas Health Science Center at Houston.

Mutagenesis of the cbb3-Type Oxidase from V. cholerae. Site-directed mutagenesis

was performed using Stratagene QuikChange kits as previously reported [20]. The

quick change PCR primers used for mutagenesis were synthesized by Integrated DNA

Technologies (IDT). Sequence verification of the mutagenesis reactions was

performed at the Biotechnology Center at the University of Illinois,

Urbana-Champaign.

Purification of the His-tagged cbb3-Type Oxidases. The mutant cbb3 proteins from R.

sphaeroides and V. cholerae with polyhistidine tags were overexpressed and purified

as previously described [20, 30]. R. sphaeroides was grown semiaerobically at 30°C

in Sistrom media with 2 µg/mL tetracycline (Sigma). V. cholerae cells were grown

aerobically at 37°C in LB media (USB Corp.) with 100 µg/mL ampicillin (Fisher

Biotech) and 100 µg/mL streptomycin (Sigma). cbb3 oxidase overexpression in V.

cholerae was induced with 0.2% L-(+)- arabinose (Sigma). The cells were collected

by centrifugation at 7000 rpm for 15 min when their growth reached early stationary

phase. Then the cells were lysed by microfluidizer (Watts Fluidair, Inc.) and

centrifuged at 40000 rpm for 4 hours to collect the membranes. Afterwards the

membra -D-maltoside (Anatrace).

Nonsolubilized membranes were removed by centrifuging at 40000 rpm for 30 min.

The protein was then purified using a nickel affinity column (Qiagen, CA) and eluted

using a stepped gradient of imidazole.

Heme Analysis of Proteins. Heme staining was used to identify subunits II and III,

CcoO and CcoP, containing covalently attached heme c [35]. GeneMate Express

PAGE gels from ISC BioExpress were used to separate the purified protein complexes.

The gels were then incubated in 3 parts 6.3 mM 3,3’,5,5’-tetramethylbenzidine

28

(TMBZ from Sigma) and 7 parts 0.25 M sodium acetate, pH 5.0, for 1 h. The gels

were then stained for heme c by adding H2O2 to 30 mM.

Spectroscopic Analysis. Spectra were acquired using Shimadzu UV/Vis-2101PC

spectrophotometer. The concentrated protein samples were diluted with 50mM

sodium phosphate buffer and 0.05% DDM at pH 6.5. The enzymes were oxidized

with 50µM Fe(CN)6 and reduced with sodium dithionite (Sigma). Spectra were

measured from 300 to 800 nm and analyzed using Origin.

Pyridine Hemochrome Spectra Assay. 0.5ml of a stock solution containing 200mM

NaOH and 40% (by volume) pyridine and 3µl of 0.1M K3Fe(CN)6 were placed in a

1ml cuvette. A 0.5ml aliquot of the protein sample (~5mM) was added with thorough

mixing and oxidized spectrum was recorded within 1 min. Solid sodium dithionite

(2-5mg) was then added and several successive spectra of the reduced pyridine

hemochromes were recorded. The absorbance differences at the selected wavelengths

were multiplied as a vector by the inverse of the matrix of extinction coefficients at

these wavelengths to obtain the concentration of heme b and heme c.

Steady-state Activity Measurements. A YSI model 53 oxygen monitor was used to

polarographically measure steady-state oxidase activity at 25°C. 1.8 mL of 50 mM

sodium phosphate and 100 mM NaCl buffer at pH 6.5 containing 0.05% DDM was

mixed with 10µL 1 M ascorbate, pH 7.4, and 20µL of 0.1 M TMPD in the sample

chamber. Horse heart cytochrome c (Sigma) was added as the substrate to a final

concentration of 40 µM. The reaction was initiated by adding 10 µL of 1 µM enzyme,

and oxygen consumption was monitored. The enzyme turnover was calculated based

on the slope of the oxygen-consumption traces.

29

2.4 Results

Mutagenesis of heme c binding sites in CcoO and CcoP

All the conserved heme c binding motifs CXXCH in CcoO and CcoP are

changed by site-directed mutagenesis of cystine or histidine to other amino acids to

determine the importance of these conserved residues. In R. sphaeroides single

mutations of His-72 from CcoO and His-126 and His-223 from CcoP were made to

alanine. Double mutations of Cys-68/Cys-70 from CcoO and Cys-122/Cys-125 and

Cys-219/Cys-222 from CcoP were changed to serine. Whereas in V. cholerae single

residues Cys-70 from CcoO and Cys-168 and Cys-253 from CcoP were substituted by

serine. Triple mutant of the binding motif Cys-70/Cys-73/His-74 from CcoO was

made as C70S/C73S/H74A to totally remove the heme c. In subunit CcoP Cys-168

and Cys-253 from the two different heme c binding motifs were mutated to serine

simultaneously to check the consequence of the disturbance of both of heme c binding

environment in CcoP (Table 1). All plasmids carrying the different mutations were

transformed into the Δcbb3 mutant strains for overexpression.

Heme c binding sites in CcoO are required for the functional cbb3 oxidase

It has been demonstrated that CcoO subunit is necessary for the stability and

assembly of cbb3 oxidase from R. sphaeroides [28]. Here we tested the importance of

the heme c binding sites in subunit CcoO from R. sphaeroides and V. cholerae. All the

His-tagged mutant proteins were purified for biochemical characterization.

SDS-PAGE gel showed that the single residue mutant H72A (R. sphaeroides) and

C70S (V. cholerae) contained the assembled subunits as like in wildtype protein.

Heme staining analysis also indicated the amounts of heme c in CcoO and CcoP

subunits were not changed (Fig. 1). Pyridine hemochrome spectra assay showed that

the heme c / heme b ratios were 3:2 and all the features were quite similar to

30

wild-type enzyme (Table 2). All the evidences indicate that single mutation of the

heme c binding sites has no effect of the heme c incorporation and assembly of the

protein complex. Using horse heart cytochrome c as the substrate protein turn over

rates were monitored for the purified cbb3 oxidase mutants compared with the

wild-type enzyme. The single residue mutant H72A (R. sphaeroides) from CcoO

subunit caused more than 90% decrease of cytochrome c oxidase activity compared

with wild-type enzyme. But the cysteine mutant C70S from V. cholerae showed

around half of the wild-type activity (Table 1). This could be due to the effect of

different residues in different stains.

However, the double-residue mutant C68S/C70S from R. sphaeroides and

triple-residue mutant C70S/C73S/H74A from Vibrio cholerae showed no assembly of

the protein complex at all. No proteins could be purified by HIS-tag column. The

elution solution was analyzed by spectrometry and SDS-PAGE gel with heme staining

which showed no heme b or c could be detected (Fig. 1). These results suggest that

heme c is coordinated by several amino acids and the disturbance of one site cannot

eliminate the binding of heme c from where electrons can still be transferred to the

binuclear center. But once all the conserved residues in the binding motif are mutated,

heme c will be lost and the protein complex could not be assembled. Heme c in CcoO

is not only required for the functionality of the cbb3 oxidase but also necessary for the

assembly and maturation of cbb3 protein complex.

Heme binding sites for the two heme c in CcoP are all required for the functional

cbb3 oxidase

Currently it’s not clear why the CcoP subunit requires diheme incorporation

and there is no direct evidence to show if both of the heme c are involved in the

mediation of the electron transfer to the catalytic subunit CcoN. To investigate the

31

role of the hemes in CcoP we performed series of mutations on the conserved heme c

binding sites. All of the mutant proteins were purified by passing through

nickel-affinity columns. The single histididine mutant H223A from R. sphaeroides

showed around one tenth of the wild-type activity while C168S and C253S mutants V.

cholerae from showed around one third and one fifth activity respectively compared

with the wild-type enzyme (Table 1). By performing spectrometry and SDS-PAGE gel

analysis it was shown that all of these mutants maintained the integrity of the subunits

and cofactors like heme b and heme c. Among all the mutants only the histidine

mutant H126A from R. sphaeroides showed significant differences in respect of the

subunits and heme contents. SDS-PAGE gels showed the subunit CcoP was missing

while CcoN and CcoO subunits were present (Fig. 1). Heme staining assay and

reduced minus oxidized difference spectra analysis indicated the loss of heme c (Fig.

2). This histidine mutant resulted in the CcoN-CcoO sub-complex which showed no

activity at all. It’s similar with the result we got from CcoO that the binding sites of

both of the heme c in CcoP are essential for the activity of cbb3 oxidase and most of

the single residue mutations are not enough to get rid of the hemes except H126A (R.

sphaeroides). The double residue mutants C122S/C125S and C219S/C222S from R.

sphaeroides showed no activity and no assembly of the cbb3 oxidase complex. And

more interestingly even if the two cysteines from the different binding motifs were

mutated the protein complex could not be assembled as shown in the double-residue

mutants H126A/H223A from R. sphaeroides and C168S/C253S from V. cholerae

strain. So we conclude that if one heme c is lost or both heme c binding sites are

perturbed the protein complex assembly process is altered and could not happen.

The conserved Histidine residues in CcoN for binding heme b and copper are

critical for the activity of cbb3 oxidase from R. sphaeroides

32

His-405 (R. sphaeroides) was predicted to be the ligand of high spin heme b3

[25]. When this histidine was mutated to alanine, no cbb3 protein could be detected or

purified from His-tag column which indicated the assembly defect. The highly

conserved His-267 was believed to be one of three ligands binding CuB and act as the

cross-link factor of His-Tyr [36]. In current study this histidine was altered to alanine

which also resulted in assembly defect. The flow-through and elution solution from

Nickel-affinity column showed no cbb3 enzyme. There was no turn over detected by

oxygen electrode. Reduced minus oxidased spectra of the elution indicated there were

no heme b or heme c.

His-407 was believed to be one of the two ligands of low spin heme b. The

mutation of this residue to alanine led to big decrease of the activity (7%) compared

with the wild-type enzyme (Table 1). All the subunits were present in SDS-PAGE gel

as like the wild-type cbb3 oxidase (Fig. 1). The dithionite-reduced minus air-oxidized

spectrum of this mutant didn’t show the 561 nm peak which is the diagnostic of

reducible heme b (Fig. 2). But the pyridine hemochrome spectroscopy analysis

showed the correct amount of heme b compared with heme c (Table 2). The protein

complex could still assemble well which suggest one histidine ligand mutation is not

enough to remove heme b entirely. This histidine ligand is required for correctly

placing heme b into the oxidase so that electron flow can take place.

2.5 Discussion

It has been well demonstrated that the biogenesis of cbb3–type oxidase

requires all the cofactors to be pre-recruited into the enzyme [34, 37]. The mutations

resulting loss of any of the metal groups brought the assembly defects of this protein

33

complex. Those cofactors include the high spin heme b3, low spin heme b and copper

from subunit CcoN and all heme c from CcoO and CcoP. In the current work,

site-directed mutagenesis studies were applied to prove that those cofactors were also

essential for the stability and assembly of cbb3–type oxidases from R. sphaeroides and

V. cholerae.

The single residue mutants H72A (R. sphaeroides) and C70S (V. cholerae)

from subunit CcoO showed only substantial decrease of activity but retained heme c

and all the functional subunits. It seems as long as the binding motif CXXCH contains

most intact binding residues heme c could still be incorporated into the enzyme

somehow. But as shown in the double-residue mutant C68S/C70S (R. sphaeroides)

and triple- residue mutant C70S/C73S/H74A (V. cholerae) cbb3–type oxidases could

not be detected which suggests that severe disturbance of the heme c binding sites is

deleterious to the attachment of heme and therefore the formation of enzyme complex.

This is consistent with the earlier results reported for cbb3–type oxidase from

Bradyrhizobium japonicum [34].

With no exception subunit CcoP of all the cbb3–type oxidases characterized

contains double heme C which could transfer the electrons from bc1-complex to

catalytic subunit CcoN. There is no united explanation why this subunit is required in

the cbb3 enzyme complex. As shown in B. japonicum [27] and Paracoccus

denitrificans [26] subunits CcoN and CcoO could assemble well with functional

catalytic activity without the incorporation of subunit CcoP. But in R. sphaeroides [38]

and Rhodobacter capsulatus [29] no active CcoN-CcoO subcomplex could be

identified which indicates that subunit CcoP is essential for the stability or assembly

of the functional cbb3 protein complex. It has been also proposed that this subunit

could serve a gas-sensing part which might sense the environment change and

34

transduce the signal to electron flow [3]. Here in the present work, mutagenesis

studies of the binding motifs of both of the heme c in CcoP of cbb3 oxidases from R.

sphaeroides and V. cholerae suggest that either of the heme C plays an important role

of the biogenesis of CcoP and cbb3 protein complex. It’s not simply that this subunit

could provide an alternate route for electron transfer other than from CcoO. All the

heme C from CcoO and CcoP must cooperate somehow in a consequential manner for

the electron delivery to CcoN. As shown in the mutant H126A (R. sphaeroides)

inactive sub-complex CcoN-CcoO was detected. Therefore we propose the electrons

must be first accepted from CcoP to CcoO then to the binuclear centre in CcoN. The

reason why CcoN-CcoO could form only in this mutant is not clear. One possibility is

that the mutant CcoP subunit assisted the assembly of CcoN and CcoO and was

degraded subsequently. Note that this phenomenon was also observed in R. capsulatus

strain where only one CcoP mutant contained the sub-complex CcoN-CcoO [29].

With no active mutant proteins containing mono heme c in CcoP we have no way to

uncover the sequence of the electron transfer from one heme c to another within CcoP.

Both of the H267A and H405A mutants turned out to be essential for the

assembly and activity of cbb3–type oxidases from R. sphaeroides which is consistent

with previous report from the same strain [28]. As His-405 is the only binding ligand

of high spin heme b it’s not a surprise to see that the active site could not form and the

protein complex could not assemble together. His-267 also plays an important role as

the ligand of CuB and one of the partners in the cross-link. However, in B. japonicum

[37] it has been reported the corresponding histidine mutant leads to fully assembled

although inactive enzyme which contains full amount of copper as wildtype protein.

This discrepancy might be due to the different cbb3 oxidases in different host strain.

But the mutant H407A from both of the strains showed similar pattern that it

35

contained normal amount of heme b and decreased catalytic activity. Although

His-407 provides the binding site for low spin heme b, it’s not required for the

incorporation of this cofactor. Nevertheless this histidine is required for the correct

conformation of cbb3–type oxidase to function normally. This mutation provides a

good candidate to study the steady cycle as it prevents dithionite to reduce heme b.

36


Table 2.1: Characteristics of mutant cbb3 oxidases

R. sphaeroides V. cholerae

Location Activity(%)a Assembly

b Activity(%)

a Assembly

b

WT 100 + WT 100 +

CcoO H72A 9 + C70S 45 +

C68S/C70S 0 - C70S/C73S/H74A 0 -

CcoP H126A 0 + C168S 17 +

H223A 8 + C253S 31 +

H126A/H223A 0 - C168S/C253S 0 -

C122S/C125S 0 -

C219S/C222S 0 -

CcoN H267A 0 -

H405A 0 -

H407A 7 +

a Steady state oxidase turnover number measured as described in the text. The activity

of the wild-type cbb3 from R. sphaeroides is about 800 e-/sec/enzyme. And the

activity of the wild-type cbb3 from V. cholerae is about 500 e-/sec/enzyme. Ascorbate

and TMPD were used as the reductants together with horse heart cytochrome c as the

substrate.

b The assembly determination was based on the spectroscopy and SDS-PAGE gel

analysis of the purified proteins.

37

Table 2.2: Heme c: heme b ratio analysis of mutant cbb3 oxidases by pyridine

hemochrome spectroscopy


Location heme c / heme b heme c / heme b

WT 3/2 WT 3/2

CcoO H72A 3/2 C70S 3/2

CcoP H126A 1/2 C168S 3/2

H223A 3/2 C253S 3/2

CcoN H407A 3/2

38

Figure 2.1

A B190 kDa -120 kDa -

85 kDa -

60 kDa -

50 kDa -

40 kDa -

25 kDa -

20 kDa -

15 kDa -

CcoN

CcoP

CcoO

CcoP

CcoO

M M1 2 3 4 5 1 2 3 4 5

CcoN

CcoP CcoP

CcoO CcoO

75 kDa -

25 kDa -

20 kDa -

50 kDa -

37 kDa -

100 kDa -

150 kDa -

C D

M 1 2 3 4 M 1 2 3 4

Fig. 2.1. (A) SDS-PAGE pattern of the purified R. sphaeroides cbb3–type oxidases


stained with Coomassie Brilliant Blue R-250. Subunits CcoN, CcoO, CcoP were

indicated on the right. Lane M stands for standard marker. Lane 1 (wild type), lane 2

(H72A), lane 3 (H126A), lane 4 (H223A), lane 5 (H407A). (B) The same PAGE gel

as (A) was stained with TMBZ to detect Heme c. (C) SDS-PAGE pattern of the

purified V. cholerae cbb3–type oxidase (10 μg). Lane 1 (wild type), lane 2 (C70S),

lane 3 (C168S), lane 4 (C253S). (D) The same PAGE gel as (C) was stained with

TMBZ to detect Heme c.

39

Figure 2.2

A B

Fig. 2.2. Reduced minus oxidized difference spectra of cbb3 oxidases. (A)

Difference spectra of R. sphaeroides cbb3–type oxidases. The enzymes were oxidized

naturally by air and reduced by dithionite. The 551 peak is the indication of

cytochrome c and 561 peak is the indication of cytochrome b. (B) Difference spectra

of V. cholerae cbb3–type oxidases.

40

2.7 References

[1] Branden, G., Gennis, R. B., and Brzezinski, P. (2006) Transmembrane proton

translocation by cytochrome c oxidase, Biochim Biophys Acta 1757, 1052–1063.

[2] Hemp, J., and Gennis, R. B. (2008) Diversity of the heme-copper superfamily in

archaea: insights from genomics and structural modeling, Results Probl. Cell. Differ.

45, 1–31.

[3] Pitcher, R. S., and Watmough, N. J. (2004) The bacterial cytochrome cbb3

oxidases, Biochim Biophys Acta 1655, 388–399.

[4] Pereira, M. M., Santana, M., and Teixeira, (2001) M. A novel scenario for the

evolution of haem-copper oxygen reductases, Biochim Biophys Acta 1505, 185–208.

[5] Pereira, M. M., Sousa, F. L., Verissimo, A. F., and Teixeira, M. (2008) Looking for

the minimum common denominator in haem-copper oxygen reductases: towards a

unified catalytic mechanism, Biochim Biophys Acta 1777, 929–934.

[6] Preisig, O., Zufferey, R., Thöny-Meyer, L., Appleby, C. A., and Hennecke, H.

(1996) A high affinity cbb3-type cytochrome oxidase terminates the symbiosis specific

respiratory chain of Bradyrhizobium japonicum, J. Bacteriol. 178, 1532– 1538.

[7] Brzezinski, P., and Gennis, R. B. (2008) Cytochrome c oxidase: Exciting progress

and remaining mysteries, J Bionenerg Biomembr 40, 521–531.

[8] Gennis, R. B. (1998) Multiple proton-conducting pathways in cytochrome oxidase

and a proposed role for active-site tyrosine, Biochim Biophy. Acta 1365, 241–248.

[9] Gennis, R. B. (2004) Coupled Proton and Electron Transfer Reactions in

Cytochrome Oxidase, Front. Biosci. 9, 581-591.

[10] Chang, H. Y., Hemp, J., Chen, Y., Fee, J. A., and Gennis, R. B. (2009) The








41

Acad. Sci. U.S.A. 106, 16169-16173.

[11] Hemp, J., Han, H., Roh, J. H., Kaplan, S., Martinez, T. J., and Gennis, R. B.

(2007) Comparative genomics and site-directed mutagenesis support the existence of

only one input channel for protons in the C-family (cbb3 oxidase) of heme-copper

oxygen reductases, Biochemistry 46, 9963-9972.

[12] Kannt, A., Soulimane, T., Buse, G., Becker, A., Bamberg, E., and Michel, H.

(1998) Electrical current generation and proton pumping catalyzed by the ba3-type

cytochrome c oxidase from Thermus thermophilus, FEBS Lett. 434, 17–22.

[13] Arslan, E., Kannt, A., Thöny-Meyer, L., and Hennecke, H. (2000) The

symbiotically essential cbb3-type oxidase of Bradyrhizobium japonicum is a proton


[14] Qin, L., Hiser, C., Mulichak, A., Garavito, R. M., and Ferguson-Miller, S. (2006)

Identification of Conserved Lipid/Detergent-Binding Sites in a High-Resolution

Structure of the Membrane Protein Cytochrome c Oxidase, Proc. Natl. Acad. Sci.

U.S.A. 103, 16117-16122.

[15] Shinzawa-Itoh, K., Aoyama, H., Muramoto, K., Terada, H., Kurauchi, T.,

Tadehara, Y., Yamasaki, A., Sugimura, T., Kurono, S., Tsujimoto, K., Mizushima, T.,

Yamashita, E., Tsukihara, T., and Yoshikawa, S. (2007) Structures and physiological

roles of 13 integral lipids of bovine heart cytochrome c oxidase, EMBO J. 26, 1713–

1725

[16] Ostermeier, C., Harrenga, A., Ermler, U., and Michel, H. (1997) Structure at 2.7

Å Resolution of the Paracoccus denitrificans Two-Subunit Cytochrome c Oxidase

Complexed with an Antibody Fv Fragment, Proc. Natl. Acad. Sci. U.S.A. 94,

10547-10553.

[17] Buse, G., Soulimane, T., Dewor, M., Meyer, H. E., and Blüggel, M. (1999)

42

Evidence for a Copper-Coordinated Histidine-Tyrosine Cross-Link in the Active Site

of Cytochrome Oxidase, Protein Sci. 8, 985-990.

[18] Hemp, J., Robinson, D. E., Ganesan, K. B., Martinez, T. J., Kelleher, N. L., and

Gennis, R. B. (2006) Evolutionary Migration of a Post-Translationally Modified

Active-Site Residue in the Proton-Pumping Heme-Copper Oxygen Reductases,


[19] Rauhamaki, V., Baumann, M., Soliymani, R., Puustinen, A., and Wikstrom, M.

(2006) Identification of a histidine-tyrosine cross-link in the active site of the

cbb3-type cytochrome c oxidase from Rhodobacter sphaeroides, Proc. Natl. Acad. Sci.

U.S.A. 103, 16135-16140.

[20] Hemp, J., Christian, C., Barquera, B., Gennis, R. B., and Martinez, T. J. (2005)

Helix Switching of a Key Active-Site Residue in the Cytochrome cbb3 Oxidases,


[21] Preisig, O., Anthamatten, D., and Hennecke, H. (1993) Genes for a

microaerobically induced oxidase complex in Bradyrhizobium japonicum are essential

for a nitrogen-fixing endosymbiosis, Proc. Natl. Acad. Sci. U. S. A. 90, 3309-3313.

[22] Preisig, O., Zufferey, R., and Hennecke, H. (1996) The Bradyrhizobium

japonicum fixGHIS genes are required for the formation of the high affinity cbb3-type

cytochrome oxidase, Arch. Microbiol. 165, 297– 305.

[23] Koch, H. G., Winterstein, C., Saribas, A. S., Alben, J. O., and Daldal, F. (2000)

Roles of the ccoGHIS gene products in the biogenesis of the cbb3-type cytochrome c

oxidase, J. Mol. Biol. 297, 49–65.

[24] Kulajta, C., Thumfart, J. O., Haid, S., Daldal, F., and Koch, H. G. (2006)

Multi-step assembly pathway of the cbb3-type cytochrome c oxidase complex, J Mol

Biol. 355, 989-1004.

43

[25] Toledo-Cuevas, M., Barquera, B., Gennis, R. B., Wikström, M., and



421–434.

[26] de Gier, J. W., Schepper, M., Reijnders, W. N., van Dyck, S. J., Slotboom, D. J.,

Warne, A., Saraste, M., Krab, K., Finel, M., Stouthamer, A. H., van Spanning, R. J.,

van der Oost, J. (1996) Structural and functional analysis of aa3-type and cbb3-type

cytochrome c oxidases of Paracoccus denitrificans reveals significant differences in

proton pump design, Mol. Microbiol. 20, 1247–1260.

[27] Zufferey, R., Preisig, O., Hennecke, H., and Thöny-Meyer, L. (1996) Assembly

and function of the cytochrome cbb3 oxidase subunits in Bradyrhizobium japonicum,

J. Biol. Chem. 271, 9114–9119.

[28] Oh, J. I., and Kaplan, S. (2000) Redox signaling: globalization of gene

expression, EMBO J. 19, 4237-4247.

[29] Koch, H. G., Hwang, O., and Daldal, F. (1998) Isolation and characterization of

Rhodobacter capsulatus mutants affected in cytochrome cbb3 oxidase activity, J.

Bacteriol. 180, 969-978.

[30] Oh, J. I., and Kaplan, S., Oxygen adaptation. (2002) The role of the CcoQ

subunit of the cbb3 cytochrome c oxidase of Rhodobacter sphaeroides 2.4.1., J Biol

Chem. 277, 16220-16228.

[31] Peters, A., Kulajta, C., Pawlik, G., Daldal, F., and Koch, H. G. (2008) Stability of

the cbb3-type cytochrome oxidase requires specific CcoQ-CcoP interactions, J

Bacteriol. 190, 5576-86.

[32] Ritz, D., Thöny-Meyer, L., and Hennecke, H. (1995) The cycHJKL gene cluster

plays an essential role in the biogenesis of c-type cytochromes in Bradyrhizobium

44

japonicum, Molecular and General Genetics 247, 27-38.

[33] Fabianek, R. A., Huber-Wunderlich, M., Glockshuber, R., Kunzler, P., Hennecke,

H., and Thöny-Meyer, L. (1997) Characterization of the Bradyrhizobium japonicum

CycY protein, a membrane-anchored periplasmic thioredoxin that may play a role as a

reductant in the biogenesis of c-type cytochromes, Journal of Biological

Chemistry 272, 4467-4473.

[34] Zufferey, R., Hennecke, H., and Thöny-Meyer, L. (1997) Heme C incorporation

into the c-type cytochromes FixO and FixP is essential for assembly of the

Bradyrhizobium japonicum cbb3-type oxidase, FEBS Letters 412, 75-78.

[35] Thomas, P. E., Ryan, D., and Leven, W. (1976) An improved straining procedure

for the detection of the peroxidase activity of cytochrome P-450 on sodium dodecyl

sulfate polyacrylamide gels, Anal. Biochem. 75, 168-176.

[36] Rauhamaki, V., Baumann, M., Soliymani, R., Puustinen, A., and Wikstrom, M.

(2006) Identification of a histidine-tyrosine cross-link in the active site of the

cbb3-type cytochrome c oxidase from Rhodobacter sphaeroides, Proc. Natl. Acad. Sci.

U.S.A. 103, 16135-16140.

[37] Zufferey, R., Arslan, E., and Thöny-Meyer, L. (1998) How replacements of the

12 conserved histidines of subunit I affect assembly, cofactor binding, and enzymatic

activity of the Bradyrhizobium japonicum cbb3-type oxidase, J. Biol. Chem. 273,

6452-6459.

[38] Oh, J. I., and Kaplan, S. (1999) The cbb3 terminal oxidase of Rhodobacter

sphaeroides 2.4.1: structural and functional implications for the regulation of spectral

complex formation, Biochemistry 38, 2688-2696.

45

CHAPTER 3: COMPARATIVE GENOMICS AND SITE-DIRECTED

MUTAGENESIS SUPPORT THE EXISTENCE OF ONLY ONE INPUT

CHANNEL FOR PROTONS IN THE C-FAMILY (CBB3 OXIDASE) OF

HEME-COPPER OXYGEN REDUCTASES *

3.1 Abstract

Oxygen reductase members of the heme-copper superfamily are terminal

respiratory oxidases in mitochondria and many aerobic bacteria and archaea, coupling

the reduction of molecular oxygen to water to the translocation of protons across the

plasma membrane. The protons required for catalysis and pumping in the oxygen

reductases are derived from the cytoplasmic side of the membrane, transferred via

proton-conducting channels comprised of hydrogen bond chains containing internal

water molecules along with polar amino acid side chains. Recent analyses identified

eight oxygen reductase families in the superfamily: the A-, B-, C-, D-, E-, F-, G-, and

H-families of oxygen reductases. Two proton input channels, the K-channel and the

D-channel, are well established in the A-family of oxygen reductases (exemplified by

the mitochondrial cytochrome c oxidases and by the respiratory oxidases from

Rhodobacter sphaeroides and Paracoccus denitrificans). Each of these channels can

be identified by the pattern of conserved polar amino acid residues within the protein.

The C-family (cbb3 oxidases) is the second most abundant oxygen reductase family

after the A-family, making up more than 20% of the sequences of the heme-copper

_________________________

* Reproduced in part with permission from Hemp, J.; Han, H.; Roh, J. H.; Kaplan, S.; Martinez, T. J.;

Gennis, R. B. (2007) Comparative genomics and site-directed mutagenesis support the existence of

only one input channel for protons in the C-family (cbb3 oxidase) of heme-copper oxygen reductases.

Biochemistry 46, 9963-9972. Copyright 2007 American Chemical Society.

46

superfamily. In this work, sequence analyses and structural modeling have been used

to identify likely proton channels in the C-family. The pattern of conserved polar

residues supports the presence of only one proton input channel, which is spatially

analogous to the K-channel in the A-family. There is no pattern of conserved residues

that could form a D-channel analogue or an alternative proton channel. The functional

importance of the residues proposed to be part of the K-channel was tested by

site-directed mutagenesis using the cbb3 oxidases from R. sphaeroides and Vibrio

cholerae. Several of the residues proposed to be part of the putative K-channel had

significantly reduced catalytic activity upon mutation: T219V, Y227F/Y228F, N293D,

and Y321F. The data strongly suggest that in the C-family only one channel functions

for the delivery of both catalytic and pumped protons. In addition, it is also proposed

that a pair of acidic residues, which are totally conserved among the C-family, may be

part of a proton-conducting exit channel for pumped protons. The residues

homologous to these acidic amino acids are highly conserved in the cNOR family of

nitric oxide reductases and have previously been implicated as part of a

proton-conducting channel delivering protons from the periplasmic side of the

membrane to the enzyme active site in the cNOR family. It is possible that the

C-family contains a homologous proton-conducting channel that delivers pumped

protons in the opposite direction, from the active site to the periplasm.

3.2 Introduction

The heme-copper superfamily is structurally and catalytically diverse, with

members that perform either oxygen reductase or nitric oxide reductase reactions. The

oxygen reductases are terminal oxidases in the respiratory chains of mitochondria and

aerobic bacteria and archaea. These enzymes catalyze the reduction of O2 to H2O

47

utilizing a bimetallic active site that contains a high-spin heme and a copper ion. The

oxygen reductases couple the chemical reaction to an electrogenic proton pump in

which one proton is pumped across the membrane per electron transferred to the

active site (1-3). The proton electrochemical gradient (proton motive force) produced

can be coupled to chemical synthesis, membrane translocation processes, and

bacterial locomotion. The net reaction is where the subscripts IN and OUT refer to the

cytoplasm and periplasm, respectively, for the bacterial and archaeal enzymes.

O2 + 8H+

IN + 4e- =2H2O + 4H

+OUT

The nitric oxide reductases catalyze the reduction of NO to N2O at an active

site consisting of a high-spin heme and an iron ion. The nitric oxide reductases are not

electrogenic and do not pump protons or generate a protonmotive force (4). The

protons used for chemistry in the nitric oxide reductases come from the periplasm, the

opposite side of the membrane than in the oxygen reductases (5).

2NO + 2H+

OUT + 2e- = N2O + H2O

The oxygen reductases are multisubunit integral membrane complexes and have

been previously classified into three families based on structural analyses: the A-, B-,

and C-families of oxygen reductases (6). More recent work (Hemp and Gennis,

manuscript in preparation) has identified five additional oxygen reductase families:

the D-, E-, F-, G-, and H-families. It appears that enzymes from each of the eight

oxygen reductase families couple the reduction of oxygen to proton pumping and the

generation of a transmembrane voltage (7-13), whereas nitric oxide reductases from

either of the known families (cNOR and qNOR families) do not pump protons and are

not electrogenic (5, 14-16).

Subunit I is the core of the multisubunit complex and defines the heme-copper

superfamily. It is the only subunit common to all of the oxygen reductase families and

48

contains the ligands for three redox-active metals: a low-spin heme (two histidine

ligands) and the bimetallic catalytic site composed of a high-spin heme (one histidine

ligand) and a nearby copper ion, CuB (three histidine ligands). The lowspin heme

accepts electrons from the electron donor specific for the particular complex and

transfers them to the catalytic site for oxygen reduction. X-ray crystallographic

structures have been reported for members of both the A-type (17-21) and B-type (22)

oxygen reductase families. These structures show that the core of subunit I is formed

by 12 transmembrane helices arranged in a pseudo-3-fold rotational symmetry with

the symmetry axis perpendicular to the membrane. The 3-fold symmetry produces

three pores (A, B, and C) which span the length of the protein (Figure 1).

The low-spin heme is located in pore C, whereas the binuclear center is in

pore B. In members of the A- and B-families X-ray crystallography (17, 18, 21) and

mass spectrometry (23, 24) have identified a novel cross-inked histidine-tyrosine

cofactor in the active site. The cofactor is formed between a conserved active site

tyrosine and one of the histidine ligands to CuB. Recently, a similar crosslinked

cofactor has been found in the C-family of oxygen reductases (23, 25). However, in

this family the tyrosine residue involved in the cross-link is located within a different

transmembrane helix in comparison to the A- and B-families (26).

Mutagenesis studies of members of the A-family have identified two

conserved proton input channels necessary for function: the D-channel and the

K-channel (27-31). The K-channel (located in pore B, Figure 1) leads from a glutamic

acid residue on the cytoplasmic side of the membrane, near the interface of subunits I

and II, to the conserved His-Tyr cofactor in the active site (32, 33). The K-channel is

responsible for the delivery of at least one, and likely two, of the protons used in

catalysis to form H2O (34). The D-channel (located in pore A, Figure 1) leads from an

49

aspartate on the cytoplasmic surface of subunit I to a proton accepting residue (a

glutamate in some A-type subfamilies or a tyrosine in others) near the active site. This

channel has been implicated in the transfer of both catalytic protons and protons

which are pumped across the membrane (34). A third proton-conducting channel

(H-channel) has been proposed on the basis of the structure of the bovine cytochrome

c oxidase (17, 35-37); however, strong supporting evidence for a functional role is

lacking, and the putative pathway is not found in the microbial A-type oxygen

reductases (38, 39).

Both the D- and K-channels contain highly conserved polar and ionizable

residues that guide the formation of hydrogen-bonded water chains, assuring

kinetically competent proton transfer (21, 40, 41). The polar and ionizable amino acid

side chains also can electrostatically stabilize the transferred proton along the water

chain within the channel (42, 43). Without the electrostatic stabilization provided by

the protein, protons could be excluded from water-containing pores (40).

The residues which form the proton channels in the A-family are not conserved in the

C-family of oxygen reductases. Since the C-type oxygen reductases have been shown

to pump protons (9, 44-47), it is certain that there must be proton-conducting channels

in this family that play analogous roles to the D- and/or K-channels. In this work,

sequence analyses and structural modeling are used to identify residues which might

form putative proton channels in the C-family of oxygen reductases. The functional

importance of the identified residues was then examined by site-directed mutagenesis

of the cbb3-type respiratory oxidases from Vibrio cholerae and Rhodobacter

sphaeroides. The data support the proposal that the C-type oxygen reductases have a

proton-conducting channel that is spatially analogous to the K-channel in the A-type

oxygen reductases. However, there are no conserved hydrophilic residues in the

50

C-type oxidases that could form either a channel analogous to the D-channel or an

alternative channel from the cytoplasmic surface. Hence, in the C-type oxygen

reductases, it is suggested that only one channel is used for the input of both catalytic

and pumped protons. In addition, a possible proton exit pathway for pumped protons

in the C-type oxygen reductases is proposed on the basis of homology to a putative

proton input channel in the cNOR nitric oxide reductase family (14).


Sequence Analysis. DNA sequences from over 850 prokaryote genomes and 9

metagenomic projects were analyzed for the presence of C-type oxygen reductase

genes using BLAST (48). Supporting Information Table S1 lists the genome and

metagenomic sequences analyzed along with their accession information. Sequence

alignments were performed using MUSCLE 3.52 (49) and manually adjusted with

Jalview (50). Transmembrane regions of the C-type oxygen reductase family were

predicted from multiple sequence alignments using TMAP (51).

Homology Modeling. Structural models of subunit I for various members of the

C-type oxygen reductase family were generated using homology modeling techniques

as described in Hemp et al. (26). Swiss-Model (52) was used to generate homology

models based on crystal structures of members of the heme-copper superfamily. The

high-spin heme, lowspin heme, and copper ion were incorporated into the modeled

protein structures, and water molecules were predicted using DOWSER (53).

Minimization of the models was performed with NAMD2 (54) using a quenched MD

simulated annealing procedure. A modified version of the CHARMM27 Proteins and

Lipids release force field (55) that included modified parameters for heme b and CuB

(26) was used in the calculations.

51

Mutagenesis of the C-Type Oxygen Reductase from V. cholerae. The oligonucleotides

used for mutagenesis were synthesized by IDT. Site-directed mutagenesis was

performed using Stratagene QuikChange kits as previously reported (26). Sequence

verification of the mutagenesis reactions was performed at the Biotechnology Center

at the University of Illinois, Urbana-Champaign.

Mutagenesis of the C-Type Oxygen Reductase from R. sphaeroides. The expression

vector pUI2803NHIS (56) was used for site-directed mutagenesis. pUI2803NHIS

encodes subunit I with a C-terminal polyhistidine tag. Point mutations of subunit I

(fixN) were made by recombinant PCR and then subcloned into pUI2803NHIS.

Sequence verification of the mutagenesis products was performed at the Molecular

Genetics Core Facility, Department of Microbiology and Molecular Genetics, The

University of Texas Health Science Center at Houston.

Purification of Recombinant Proteins. The mutant proteins with polyhistidine tags

were overexpressed and purified as previously described (26). Briefly, V. cholerae

cells were grown at 37 C in LB media (USB Corp.) with 100 μg/L ampicillin (Fisher

Biotech) and 100 μg/L streptomycin (Sigma). Protein expression was induced with

0.2% L-(+)-arabinose (Sigma). R. sphaeroides was grown semiaerobically at 30 C

in Sistron media with 2 mg/L tetracycline. The cells were lysed and centrifuged at

40000 rpm to collect the membranes. The membranes were solubilized with 0.5%

dodecyl â-D-maltoside (Anatrace). Nonsolubilized membranes were removed by

centrifuging at 40000 rpm for 30 min. The protein was then purified using a nickel

affinity column (Qiagen, CA) and eluted using a stepped gradient of imidazole.

Heme Analysis of Proteins. Heme staining was used to identify subunits II and III,

CcoO and CcoP, containing covalently attached heme c (57). GeneMate Express


52

The gels were t -tetramethylbenzidine

(TMBZ from Sigma) and 7 parts 0.25 M sodium acetate, pH 5.0, for 1 h. The gels

were then stained for heme c by adding H2O2 to 30 mM.

Spectroscopic Analysis. Spectra were acquired using an Agilent Technologies 8453

UV-visible spectrophotometer running ChemStation software. Twenty-five microliters

of the protein sample at 130 mM was mixed with 100 μL of 50 mM sodium phosphate

buffer and 0.05% DM at pH 6.5. The enzymes were oxidized with 2 μL of 1 mM

Fe(CN)6 and reduced with dithionite, both obtained from Sigma. Spectra were

measured from 375 to 850 nm and analyzed using Matlab.

Oxidase ActiVity Measurements. A YSI model 53 oxygen monitor was used to

polarographically measure steady-state oxidase activity. The buffer used for oxidase

activity measurements was 50 mM sodium phosphate and 50 mM NaCl at pH 6.5. Ten

microliters of 1 M ascorbate, pH 7.4, and 18 μL of 0.1 M TMPD were mixed with 1.8

mL of buffer in the sample chamber at 25 C. The reaction was initiated by adding 10

μL of 1 μM enzyme, and oxygen consumption was monitored.

3.4 Results

Genomic and sequence analyses, structural modeling, and mutagenesis were

performed to investigate the properties of proton channels in the C-type oxygen

reductase family. Evolutionarily conserved residues were mapped onto structural

models of the C-type family in order to identify potential proton channels.

Mutagenesis studies were then performed to verify the roles of the predicted residues

in channel formation.

Sequence and Structural Analysis of the C-Family of Oxygen Reductases. DNA

sequences from over 850 prokaryotic genomes, nine metagenomic projects, and

53

individually submitted sequences to Genbank were analyzed for members of the

C-type oxygen reductase family. BLAST analysis identified 380 (275 genomic/90

metagenomic/15 individual submissions) putative members of the C-type family

(Supporting Information Table S2). The 275 genomic members represent 23.5% of the

total number of oxygen reductases and 20.7% of the total number of heme-copper

superfamily members identified. The C-type family has a broad phylogenetic

distribution with members from sequenced genomes being unevenly distributed

among 14 of the 23 officially named bacterial phyla currently recognized by the

NCBI Taxonomy Database. [It should be noted that there are likely to be over 100

bacterial phyla, many of which are not represented in the NCBI Taxonomy Database

(58).] The majority (89%) were found within the proteobacteria (Supporting

Information Table S2); however, the skewed phylogenetic distribution toward

proteobacteria is clearly due to the disproportionate genomic sequencing efforts on

members of this phylum. To date no members of the C-type oxygen reductase family

have been found in archaea or eukaryotes. A complete genomic and phylogenetic

analysis along with subfamily classification of the C-type oxygen reductase family

will be presented elsewhere.

Sequence alignments were used to identify conserved amino acid residues

which may play important functional roles in members of the C-type oxygen

reductase family (Supporting Information Figure S1). Table 1 lists the completely

conserved (>99%) and partially conserved (>95%) residues within the family. The

conserved residues are classified into four categories: (1) residues conserved in all

members of the superfamily; (2) residues conserved within all of the oxygen reductase

families but not the NO reductases; (3) residues conserved between the C-type oxygen

reductases and at least one of the other families within the superfamily (either oxygen

54

or nitric oxide reductases); and (4) residues conserved only within the C-type oxygen

reductase family.

(1) There are only five residues completely conserved in all members of the

superfamily. These five residues are the histidines that ligate the low-spin heme (two

histidines), highspin heme (one histidine), and active site metal ion (two histidines),

which is copper, CuB, in the oxygen reductases or an iron, FeB, in the nitric oxide

reductases. The active site metal ion is usually ligated by three histidines; however, a

new heme-copper family has been found in which one of the histidines is replaced by

an aspartate (to be described elsewhere).

(2) There are no residues uniquely conserved in all of the oxygen reductases that are

not also conserved within the nitric oxide reductases. This implies that for functions

that are unique to the oxygen reductases, such as proton pumping, different amino

acids may play the same structural/functional role in different oxygen reductase

families.

(3) There are numerous residues conserved between the C-type family of oxygen

reductases and at least one other family of either oxygen reductases or nitric oxide

reductases within the superfamily (Table 1). Four residues are particularly interesting:

E126, E129, Y227, and T297 (V. cholerae numbering). Residues homologous to E126

and E129 are also conserved in the cNOR family of NO reductases, where they have

recently been shown (59) to be components of a proton input pathway from the

periplasmic surface to the active site (5, 14, 15). Y227 is homologous to a conserved

tyrosine in the B-type oxygen reductase family that has been proposed to be part of

the putative K-channel, and T297 is homologous to a conserved S/T located within

the K-channel (6) in the A-type oxygen reductases.

(4) There are ten residues with >99% conservation unique to the C family (Table 1).

55

One of these residues is a glycine (G199). Three of the residues (W166, F248, and

W249) form a conserved hydrophobic cluster and are likely to be important for

structural reasons. The other six conserved residues unique to the C-family are polar

amino acids that are located within pore B (Figure 1) and are candidates for being part

of a proton-conducting K-channel analogue: T219, Y228, S240, Y255, N293, and

Y321. Y255 is the conserved cross-linked active site tyrosine, previously identified

(26).

To identify amino acids which could form proton channels analogous to those

in the A- and B-type oxygen reductase families, we mapped conserved residues onto

structural models of members of the C-type oxygen reductase family.

We identified nine conserved residues which could form a proton channel

analogous to the K-channel: T219, Y227, Y228, S240, S244, Y255, N293, T297, and

Y321. A model of the V. cholerae C-type oxygen reductase highlighting these residues

is shown in Figure 2. Sequence alignments of the whole family show that there are no

conserved or partially conserved residues that could form a channel analogous to the

D-channel (Supporting Information Figure S1). Since the heme-copper superfamily is

ancient, it is possible that residues forming a second proton channel might only be

conserved within individual subfamilies of the C-type oxygen reductases and that

different subfamilies may have channels composed of different conserved residues.

Sequence, structural, and phylogenetic analyses have identified at least 13 subfamilies

of C-family oxygen reductases. Sequence analyses of these subfamilies revealed no

conserved residues even within subfamilies which could form a channel analogous to

the D-channel.

Site-Directed Mutagenesis of Conserved Residues Proposed To Form a Proton Input

Channel. Sequence analysis and structural modeling (described above) identified the

56

following nine conserved polar residues as likely forming a proton-conducting

channel analogous to the K-channel in the A-family of oxygen reductases: T219,

Y227, Y228, S240, S244, Y255, N293, T297, and Y321. Previously, Y255 was

demonstrated to be essential for catalytic function, and this residue was shown to be

part of the cross-linked cofactor in the enzyme active site (23, 25). The remaining

eight conserved residues in subunit I (CcoN) of the V. cholerae C-type oxygen

reductase were each replaced by site-directed mutagenesis to assess their functional

importance: T219V, Y227F/Y228F (double mutant), N293D, Y321F, S240A, S244A,

and T297V. All of the mutants were expressed and assembled correctly, as determined

by UV-vis spectroscopy and heme analysis (Figure 3). Oxygen reductase activity

assays were performed to assess the effect of each mutation on catalysis (Table 2).

Four of the mutants [T219V, Y227F/Y228F (double mutant), N293D, and Y321F] had

significantly reduced catalytic activity or were completely inactive, whereas three of

the mutations (S240A, S244A, and T297V) had no effect on oxidase activity. The

locations of some of the residues are shown in Figure 2.

Mutations were also made at four sites in the C-type oxidase from R.

sphaeroides: T275G, Y311G, N349G, and Y377G. Y311 is the active site tyrosine,

equivalent to V. cholerae Y255, and as expected, the Y311G mutant is virtually

inactive (Table 2). This has been shown previously (47). The T275G, N349G, and

Y377G mutants also have low catalytic activity, qualitatively consistent with the

results from the mutations in the equivalent sites (T219V, N293D, and Y321F) in the

V. cholerae enzyme. The influence of these three mutations in the R. sphaeroides

oxidase is much more dramatic than the mutations in the equivalent positions in the V.

cholerae oxidase (Table 2). This may be due to the fact that glycine was placed in

each position in the R. sphaeroides oxidase, which may cause structural perturbations.

57

Heme analysis of the mutants from the R. sphaeroides oxidase is consistent with

structural perturbations resulting from the glycine substitutions. Whereas the

wild-type R. sphaeroides oxidase has a measured heme c:heme b ratio of 3:2, this

ratio is altered in the mutants: T275G (2:1), Y311G (3:1), N349G (4:1), and Y377G

(4:1). This is not observed with the mutants of the V. cholerae oxidase (T219V,

N293D, Y311F, and Y321F).

Previously, H303 was proposed to be part of the K channel in the cbb3-type

oxidases (47). However, the larger data set used in the current work shows that H303

is not conserved either within the family of C-type oxygen reductases or within any of

the subfamilies. The H303G mutant in the current work (Table 2) has very low

catalytic activity (4%), and the heme c:heme b ratio is the same as the wild type,

suggesting no major problem of assembly. However, it has recently been shown (56)

that the H303A mutant in the same R. sphaeroides oxidase is fully active, indicating

that the histidine at this location is not critical for activity.

Site-Directed Mutagenesis of ConserVed Residues Proposed To Form a Proton Exit

Channel. Two acidic residues, E126 and E129 (V. cholerae), are totally conserved in

the C-family oxygen reductases and in the cNOR family of nitric oxide reductases

(Supporting Information Figure S2). Mutations were made of both glutamates in the V.

cholerae (E126Q and E129Q) and in the R. sphaeroides (E180G and E183G) C-type

oxygen reductases to determine their functional importance. The E126Q (V. cholerae)

and E180G (R. sphaeroides) mutants are virtually inactive. The V. cholerae E129Q

mutant has about 47% of the wild-type oxidase activity, whereas the equivalent R.

sphaeroides E183G mutant is virtually inactive (Table 2). The mutants in the V.

cholerae oxidase have no effect on the heme content or UVvis spectrum of the

enzyme. However, the glycine substitutions in the R. sphaeroides oxidase may have

58

caused structural effects, altering the heme ratio for E180G (2:1) and E183G (4:1).

The loss of function obtained with the V. cholerae oxidase mutants (Table 2) is

very similar to that previously published on the effects of the equivalent mutations

(E122Q and E125Q) in the NO reductase activity of a member of the cNOR family

(59). In the cNORs, the conserved glutamates are proposed to be part of a proton

input pathway (14, 59), so it is reasonable to suggest that in the C-type oxygen

reductases the two glutamates may form an exit pathway for pumped protons

delivered to the periplasm. The location of the two glutamates in a model of the V.

cholerae cbb3 oxidase is shown in Figure 4. The loop region between helix III and

helix IV in the C-type oxygen reductases is difficult to model due to the lack of

homology of this region with known crystal structures. This should be kept in mind

when proposing structural/functional features of this loop.

3.5 Discussion

Members of the heme−copper superfamily are divided into two classes: the

oxygen reductases and the nitric oxide reductases. The active sites for enzymes from

both of these classes are buried within the proteins; therefore, the protons required for

their chemistries must be provided via proton-conducting channels from the aqueous

phase. It has been demonstrated experimentally that the required protons are taken

from the inside (bacterial cytoplasm or mitochondrial matrix) for the oxygen

reductases and from the outside (bacterial periplasm) for the nitric oxide reductases.

In this work sequence analyses, structural modeling, and site-directed mutagenesis

were used to identify proton-conducting channels in the C-family of heme−copper

oxygen reductases (cbb3-type respiratory oxidases).

Previous studies have identified three distinct families of oxygen reductases (6)

59

(the A-, B-, and C-families) and two families of nitric oxide reductases (cNOR and

qNOR) (4, 60). More recently, new sequences have been made available from

microbial genome and metagenomic sequencing efforts. This has resulted in a much

larger data set for the analysis of the heme−copper superfamily, the results of which

will be published separately. There are enough sequences available to clearly define a

number of subfamilies within the A-, B-, and C-families of oxygen reductases and

also to define five additional families (D-, E-, F-, G-, and H-families). Similarly with

the nitric oxide reductases, several additional families have been identified in addition

to the cNOR and qNOR families (manuscript in preparation).

The A-family of oxygen reductases is by far the largest (72% of the 1170

sequences of heme−copper oxygen reductases included in the current analysis) and

most studied group of enzymes in the heme−copper superfamily. Several X-ray

structures have been determined and site-directed mutagenesis, along with biophysical

methods, has been extensively used to elucidate the structural and mechanistic

properties of this family. Two proton-conducting input channels, the D- and

K-channels, are well defined by both X-ray crystallography and site-directed

mutagenesis of prokaryotic members of the family (27-31). These two channels are

used to provide protons from the bacterial cytoplasm (or mitochondrial matrix) to the

active site for chemistry (4 H+) and for proton pumping (4 H

+). The D-channel has

been a focus of attention because it is clearly involved in the proton pump of the

A-family of oxygen reductases. Some mutations within the D-channel can eliminate

proton pumping (30, 61, 62) whereas other mutations completely block all catalytic

function (63). It has been noted previously that the oxygen reductases in the C-family

do not appear to have residues equivalent to those corresponding to the D-channel of

the A-family of oxygen reductases (6, 12, 47). The current work examines this using

60

the large data set now available.

The premise of the approach is that the proton-conducting channels will

involve functionally important polar and/or ionizable residues. In principle, this need

not necessarily be the case, as exemplified by the gramicidin channel, in which

backbone carbonyls are utilized to form a hydrogen bond network with water (64).

However, the gramicidin channel is different from the channels being considered in

the current work because gramicidin conducts cations other than protons whereas the

channels of interest in cytochrome oxidase only conduct protons. In channels that

only conduct protons through membrane proteins, the participation of polar/ionizable

side chains appears to be universal (41, 65). This is clearly observed in the A-family

of oxygen reductases, where the pattern of conserved polar amino acids defines both

the K- and D-channels (6).

In the A-type oxygen reductases, the K-channel leads from a surface-exposed

glutamate at the cytoplasmic surface to the active site tyrosine, which is cross-linked

to a histidine (28, 66). Besides the active site tyrosine, there are three other conserved

residues in the A-family of enzymes which line the K-channel: T/S359, K362, and

E101II (subunit II) (numbering of the R. sphaeroides aa3-type oxidase) (Figure 2A).

Similarly, there is a pattern of highly conserved residues in the D-channel in the

A-family of oxygen reductases: D132, N121, N139, N207, Y33, and E286.

Other examples of proteins in which ionizable or polar amino acids are critical

components of proton-conducting pathways are the bacterial photosynthetic reaction

center (67, 68), the F1Fo-ATP synthase (69, 70), the bc1 complex (71-74),

bacteriorhodopsin (75), succinate dehydrogenase (76), and the recently discovered

E-channel in the fumarate reductase from Wolinella succinogenes (77).

Of particular interest is the cNOR family of nitric oxide reductases. Sequence

61

analysis shows no pattern of conserved polar residues that could define a proton input

channel from the bacterial cytoplasm (Hemp and Gennis, in preparation) but does

reveal the presence of two conserved glutamates that could be involved in forming a

proton input channel from the periplasmic side of the membrane. Experimental data

confirm that protons are, indeed, taken from the periplasm and that the two glutamates

do appear to be important for this function (14, 59). Hence, within the heme−copper

superfamily, sequence alignments predicting the presence or absence of

proton-conducting channels have been validated experimentally for the D- and

K-channels of the A-family of oxygen reductases and the cNOR family of nitric oxide

reductases.

In light of these correlations, it is meaningful that the pattern of conserved

residues identified only one proton input channel for the C-family of oxygen

reductases, spatially equivalent to the K-channel in the A-family of oxygen reductases,

though largely comprised of different residues. The only residue in common between

the K-channels in the A- and C-families of enzymes corresponds to T359 in the R.

sphaeroides aa3-type oxidase. The expectation tested in this work is that mutations of

conserved, polar residues in the putative K-channel will block proton translocation

required for both chemistry and proton pumping by the C-family oxygen reductases.

The K-Channel. Five of the nine residues (apart from the active site tyrosine) that

were proposed to be part of the K-channel in the C-family of oxygen reductases were

implicated as being functionally important for catalytic function by site-directed

mutagenesis of the V. cholerae and R. sphaeroides cbb3-type oxidases: T219, Y227

and/or Y228, N293, and Y321 (V. cholerae numbers). The location of these residues

in a model of the CcoN subunit of the V. cholerae enzyme is shown in Figure 2B. In

addition, it was previously shown that the active site tyrosine (Y255), located at the

62

terminus of the K-channel is essential for function (26). In the A-family of oxygen

reductases, there is a conserved glutamic acid in subunit II which is located at the

entrance of the K-channel, E101II in the R. sphaeroides aa3-type oxidase (78). In the

C-family of enzymes, a homologue of subunit II is not present, and there are no

conserved residues in any of the accessory subunits of the C-family of oxygen

reductases (CcoO, CcoP, or CcoQ) that could be identified as equivalent to the

glutamate in the A-family of enzymes.

It is noted that the K-channel analogue in the C-type oxygen reductases

contains no protonatable residues, in contrast to both the D- and K-channels in the

A-type oxygen reductases. It has been suggested previously that a histidine residue

(H303 in the R. sphaeroides C-type oxygen reductase) near N293 might be an

important component of the K-channel in the C-type oxygen reductases (47).

Mutation of this histidine to valine resulted in reduced oxidase activity (79, 80).

However, this residue is not conserved in the C-family (Supporting Information

Figure S1), and mutation to alanine (H303A) in the R. sphaeroides cbb3-type oxidase

has been reported to have no effect on enzyme activity (56). The current work (Table

2) shows that a glycine is not tolerated at this location, which does not imply that

H303 is a required for proton translocation.

The three conserved residues for which mutation had no effect on activity

(S240, S244, and T297) (see Figure 2B) are all located just below N293 near the

entrance to the channel. The N293 residue is estimated in structural models to be

7−10 Å from the cytoplasmic surface and unlikely to have direct contact with protons

from the cytoplasm. A proton-conducting pathway should be required for the transfer

of protons from the cytoplasm to the level of N293. The S240, S244, and T297

residues could participate in this, but it may be that there are multiple pathways

63

possible so that no single residue is essential for the function. A similar situation is

proposed for the highly conserved serines and threonines (S142, S200, and S201; R.

sphaeroides aa3-type numbering) within the D-channel of the A-family of oxygen

reductases (81). Multiple mutations might resolve this matter.

Absence of a Second Proton Input Channel in the C-Family of Oxygen Reductases.

Analysis of the C-family shows that no residues are conserved that could reasonably

be postulated to be components of a proton input channel apart from the K-channel.

Earlier work noted that there is a conserved tyrosine in the C-family that is

structurally analogous to the tyrosine at the top of the D-channel in some subfamilies

of the A-type oxygen reductases (6, 47). It was previously reported that mutation of

this residue to phenylalanine in the R. sphaeroides C-type oxygen reductase (Y265F)

resulted in an enzyme with low activity; however, there was no effect on pumping

(47). Sequence analysis shows that this residue is not conserved and is a

phenylalanine in many C-type sequences (Supporting Information Figure S1).

Therefore, it is unlikely that this tyrosine plays the same role as in the A-type oxygen

reductases. If the C-family has a channel analogous to the D-channel, it is not formed

by conserved residues.

An alternative proton input channel has been suggested between D364 (R.

sphaeroides cbb3 oxidase numbering) on the cytoplasmic side and E383 (R.

sphaeroides) near the high-spin heme (47). In support of this, E383 was mutated to

glutamine, resulting in complete loss of activity (47). However, sequence analysis

shows that neither D364 nor E383 are conserved within the C-family of oxygen

reductases or within any subfamily (Supporting Information Figure S1). In many

sequences the residue equivalent to E383 is a glutamine. Structural modeling (not

shown) predicts that E383 could be as close as 4 Å from the histidine ligand to the

64

high-spin heme in the active site. Hence, mutation of this residue might modify the

midpoint potential of the high- spin heme and could explain the experimental results

(47). There are no other conserved residues between D364 and E383 which could

form the rest of a channel. There are also no conserved residues anywhere else in the

protein which could form a potential alternative proton channel in any of the

subfamilies comprising the C-family of oxygen reductases.

If a second proton input channel is present in the C-family of oxygen reductases, it

does not contain conserved polar residues.

A Possible Proton Exit Channel. The C-type oxygen reductase family is closely

related to the cNOR and qNOR nitric oxide reductase families (6, 82) and shares a

number of conserved sequence features with them that are not found in the other

oxygen reductase families (Table 1). One feature is the presence of the two conserved

glutamate residues (E126 and E129, V. cholerae numbering) located in the loop region

between helix III and helix IV on the periplasmic side of the protein (Supporting

Information Figure S2). Modeling (Figure 4) shows that the conserved glutamates in

the III−IV loop region of the C-family of oxygen reductases could be in a similar

location as the glutamates in models of the cNOR nitric oxide reductases (5, 14). It is

therefore reasonable to propose that this pair of glutamates may share a common role

in both the cNOR's and the C-type oxygen reductases. Mutations were made to test

the functional importance of these glutamates in the C-family. Activity measurements

with the V. cholerae oxidase clearly demonstrate that these two glutamates are

functionally important (Table 2). These results are very similar to those obtained in

the cNOR family (59) in which it was proposed that the two glutamates are involved

in a proton-conducting pathway from the periplasm to the enzyme active site. Possibly,

the equivalent glutamates in the C-type oxygen reductases form an exit pathway for

65

protons to the periplasm. This proposal must be further examined by analysis of

proton pumping in these mutants.

The III−IV loop region has been previously modeled in a manner that places

the two glutamates within transmembrane helix IV (47). It was proposed that residue

Y181 (R. sphaeroides C-type oxidase numbering) in the III−IV loop forms a critical

association with W263 in helix VI and that K179 in the III−IV loop interacts with the

heme propionates along with R471 in the X−XII loop. These residues were all

conserved in the data set of Sharma et al. (47). However, the current sequence

analysis with the larger data set shows that K179, Y181, W263, and R471 are not

conserved within the C-family of oxygen reductases.

3.6 Conclusions

(1) Sequence analyses, structural modeling, and site-directed mutagenesis

demonstrate that the C-type oxygen reductases have a conserved proton channel

spatially analogous to the K-channel leading from the cytoplasmic surface to the

novel cross-linked histidine−tyrosine cofactor in the active site.

(2) Extensive sequence and structural analyses have also shown that the

C-type oxygen reductases do not have a conserved channel analogous to the

D-channel and that no other residues are conserved that could form an alternative

channel from the cytoplasmic surface of the protein to the active site. These results

strongly suggest that the C-type oxygen reductases have only one proton input

channel and that the K-channel is responsible for the uptake of all protons, both for

catalysis and pumping. Although the principles behind the mechanism of proton

pumping are very likely to be the same, it is evident that the functions of residues in

the D-channel shown to be critical in the A-family of oxygen reductases must be

performed differently in the C-family of oxygen reductases. The invariant structural

66

features shared by all of the heme−copper oxygen reductases are the metal ligands

and the heme propionates. These may play specific roles in the proton pump

mechanism, as has been proposed in numerous models (83-86).

(3) Two totally conserved glutamates in the C-family of oxygen reductases

may be part of a proton exit pathway.

Further experiments, including the structural determination of a C-type oxygen

reductase, will be necessary to clarify how these enzymes function.

67


Table 3.1: Conserved Residues in the C-Family of Heme−Copper Oxygen

Reductasesa

residue structural/ functional role Superfamily conservation

>99% Conservation

H64 high-spin heme ligand conserved in all HC's

E126 proton exit conserved in NOR's and

C-family

E129 proton exit conserved in NOR's and

C-family

W166 hydrophobic cluster unique to C-family

G199 structural unique to C-family

H211 CuB ligand conserved in most HC's

T219 K-channel analogue uniquetoC-family

Y227 K-channel analogue conserved in B- and

C-families

Y228 potential hydrogen bond to

R156

unique to C-family

P231 structural conserved in B- and

C-families

S240 K-channel analogue

entrance

unique to C-family

F248 Hydrophobic cluster unique to C-family

W249 Hydrophobic cluster unique to C-family

Y255 K-channel analogue/ active

site cofactor

Unique to C-family

G259 structural Conserved in NOR's and

C-family

H261 CuB ligand Conserved in all HC's

H262 CuB ligand Conserved in all HC's

P286 structural Conserved in most HC's

N293 K-channel analogue Unique to C-family

T297 K-channel analogue

entrance

Conserved in A- and

C-families

Y321 K-channel analogue Unique to C-family

H349 low-spin heme ligand Conserved in all HC's

H351 high-spin heme ligand Conserved in all HC's

G407 structural Conserved in most HC's

R441 Salt bridge withD344 Conserved in NOR's and

C-family

G445 structural Conserved in NOR's and

C-family

95% Conservation

R59 Helix cap Unique to C-family

R61 low-spin heme propionate

hydrogen bond

Conserved in NOR's and

C-family

R87 Helix cap Unique to C-family

68

Table 3.1 continued

R156 Potential hydrogen bond to

Y228

Unique to C-family

W207 Active site Conserved in allHC's

N212 unknown Unique to C-family

S244 K-channel analogue

entrance

Unique to C-family

S280 Hydrogen bond H261 Conserved in most HC's

G328 structural, allows for

high-spin heme

Conserved in all HC's

H341 Cation binding site/

high-spin heme propionate

Conserved in most HC's

T343 Cation binding site Conserved in most HC's

Y367 unknown Partially conserved in some

HC's

P371 structural Partially conserved in some

HC's

F389 structural Partially conserved in some

HC's

W390 structural Partially conserved in some

HC's

G395 structural Unique to C-family

F427 unknown Unique to C-family

G451 structural Unique to C-family

a

The conserved residues for the C-family were determined by analysis of all 275

genomic sequences (V. cholerae numbering). The residues in bold are from published

site-directed mutagenesis studies. A likely structural and/or functional role was

assigned to the conserved residues on the basis of structural analysis and mutagenesis

studies. The conservation of the conserved residues in the C-family relative to other

members of the heme−copper superfamily is given.

69

Table 3.2: Relative Oxygen Reductase Activity of Mutant C-Type Oxygen

Reductasesa

V.cholerae R.sphaeroides

Mutant activity(%) mutant activity(%) location

WT 100 WT 100

E126Q <1 E180G <1 Exit channel

E129Q 17 E183G 1.1 Exit channel

T219V 48 T275G <1 K-channel

Y227F/Y228F 57 K-channel

S240A 100

S244A 100

H303G 4 K-channel

Y255F <1 Y311G <1 K-channel

T297V 110

N293D 26 N349G <1 K-channel

Y321F <1 Y377G <1 K-channel

S244AN293D 25

a

The residues in each row are structurally equivalent. The numbering is for the

respective sequences.

70

Figure 3.1

Fig. 3.1. Pore structure of cytochrome c oxidase. Cytochrome c oxidase from R.

sphaeroides viewed perpendicular to the membrane from the periplasmic side

showing the structure of the three pores. Each pore is formed by four transmembrane

helices (pore A, red; pore B, green; pore C, blue). The D-channel (red residues) is

located within pore A and the K-channel (green residues) within pore B.

71

Figure 3.2

Fig. 3.2. K-channel analogue in the C-family of heme−copper oxygen reductases. (A)

A-family K-channel. Residues forming the K-channel in the A-type cytochrome c

oxidase from R. sphaeroides are shown in red (Y288, T359, K362, and E101II). The

CuB ion is in yellow, and the helices forming pore B are in green (see Figure 1). (B)

C-family K-channel analogue. K-channel analogue in the C-type cbb3 oxidase from V.

cholerae. Amino acids in red are conserved residues identified by mutagenesis as

playing a role in channel formation (Y255, Y321, T219, Y227, and N293). (C)

C-family K-channel analogue rotated by 90°. Amino acids in blue are conserved

residues which individually had no effect on oxidase activity; however, they may

collectively form an entrance to the channel (S240, S244, and T297). Amino acids in

yellow are residues which are not conserved in the C-family (H247 and S287);

however, they may play a role in channel formation in the individual sequences which

contain them.

72

Figure 3.3

Fig. 3.3. (A) Reduced spectra of mutant proteins. (B) Heme-stained PAGE gel of

selected mutants of V. cholerae C-type oxygen reductase. Heme analyses of all other

mutants were identical. Lane assignments: M, marker; 1, wild type; 2, T219V; 3,

S240A; 4, N293D; 5, T297V; 6, Y321F.

73

Figure 3.4

Fig. 3.4. Potential exit pathway. The residues identified as forming a possible exit

pathway analogous to that in the cNOR family are shown in green (E126 and E129).

These residues are located in the loop between helices III and IV at the top of pore A.

Modeling of this region is very difficult due to the lack of homology with known

structures. This figure shows one possibility of the location of these important

residues. A crystal structure of a member of the C-family will be necessary to

determine their actual location.

74

3.8 References

1. Garcia-Horsman, J. A., Barquera, B., Rumbley, J., Ma, J., and Gennis, R. B.

(1994) The Superfamily of Heme-Copper Respiratory Oxidases, J. Bacteriol.

176, 5587-5600.

2. Michel, H. (1998) The Mechanism of Proton Pumping by Cytochrome c

Oxidase, Proc. Natl. Acad. Sci. U.S.A. 95, 12819-12824.

3. Wikstrom, M., and Verkhovsky, M. I. (2006) Towards the Mechanism of

Proton Pumping by the Haem-Copper Oxidases, Biochim. Biophys. Acta 1757,

1047-1051.

4. Zumft, W. G. (2005) Nitric Oxide Reductases of Prokaryotes with Emphasis

on the Respiratory, Heme-Copper Oxidase Type, J. Inorg. Biochem. 99,

194-215.

5. Reimann, J., Flock, U., Lepp, H., Honigmann, A., and Adelroth, P. (2007) A

Pathway for Protons in Nitric Oxide Reductase from Paracoccus denitrificans,

Biochim. Biophys. Acta 1767, 362-373.

6. Pereira, M. M., Santana, M., and Teixeira, M. (2001) A Novel Scenario for the

Evolution of Haem-copper Oxygen Reductases, Biochim. Biophys. Acta 1505,

185-208.

7. Wikstrom, M. (1977) Proton Pump Coupled to Cytochrome c Oxidase in

Mitochondria, Nature 266, 271-273.


(1998) Electrical Current Generation and Proton Pumping Catalyzed by the

ba3-type Cytochrome c Oxidase from Thermus thermophilus, FEBS Lett. 434,

17-22.

9. de Gier, J.-W. L., Schepper, M., Reijnders, W. N. M., van dyck, S. J.,

75

Slotboom, D. J., Warne, A., Saraste, M., Krab, K., Finel, M., Stouthamer, A.

H., van Spanning, R. J. M., and van der Oost, J. (1996) Structural and

Functional Analysis of aa3-type and cbb3- type Cytochrome c Oxidases of

Paracoccus denitrificans Reveals Significant Differences in Proton-pump

Design, Mol. Microbiol. 20, 1247-1260.

10. Gilderson, G., Aagaard, A., Gomes, C. M., Adelroth, P., Teixeira, M., and

Brzezinski, P. (2001) Kinetics of Electron and Proton Transfer during O(2)

Reduction in Cytochrome aa(3) from A. ambiValens: An Enzyme Lacking

Glu(I-286), Biochim. Biophys. Acta 1503, 261-270.

11. Gleissner, M., Kaiser, U., Antonopoulos, E., and Schafer, G. (1997) The

Archaeal SoxABCD Complex Is a Proton Pump in Sulfolobus acidocaldarius,

J. Biol. Chem. 272, 8417-8426.

12. Lemos, R. S., Gomes, C. M., Santana, M., LeGall, J., Xavier, A. V., and

Teixeira, M. (2001) The ―Strict‖ Anaerobe DesulfoVibrio gigas Contains a

Membrane-Bound Oxygen-Reducing Respiratory Chain, FEBS Lett. 496,

40-43.

13. Pereira, M. M., Verkhovskaya, M. L., Teixeira, M., and Verkhovsky, M. I.

(2000) The caa3 Terminal Oxidase of Rhodothermus marinus Lacking the

Key Glutamate of the D-Channel Is a Proton Pump, Biochemistry 39,

6336-6340.

14. Flock, U., Reimann, J., and Adelroth, P. (2006) Proton Transfer in Bacterial

Nitric Oxide Reductase, Biochem. Soc. Trans. 34, 188-190.

15. Flock, U., Watmough, N. J., and Adelroth, P. (2005) Electron/Proton Coupling

in Bacterial Nitric Oxide Reductase during Reduction of Oxygen,


76

16. Hendriks, J. H., Jasaitis, A., Saraste, M., and Verkhovsky, M. I. (2002) Proton

and Electron Pathways in the Bacterial Nitric Oxide Reductase, Biochemistry

41, 2331-2340.

17. Yoshikawa, S., Shinzawa-Itoh, K., and Tsukihara, T. (1998) Crystal Structure

of Bovine Heart Cytochrome c Oxidase at 2.8 Å Resolution, J. Bioenerg.

Biomembr. 30, 7-14.

18. Ostermeier, C., Harrenga, A., Ermler, U., and Michel, H. (1997) Structure at

2.7 Å Resolution of the Paracoccus denitrificans Two-Subunit Cytochrome c

Oxidase Complexed with an Antibody Fv Fragment, Proc. Natl. Acad. Sci.

U.S.A. 94, 10547-10553.

19. Svensson-Ek, M., Abramson, J., Larsson, G., Tornroth, S., Brzezinski, P., and

Iwata, S. (2002) The X-ray Crystal Structures of Wild-Type and EQ(I-286)

Mutant Cytochrome c Oxidases from Rhodobacter sphaeroides, J. Mol. Biol.

321, 329-339.

20. Abramson, J., Riistama, S., Larsson, G., Jasaitis, A., Svensson- Ek, M.,

Laakkonen, L., Puustinen, A., Iwata, S., and Wikstrom, M. (2000) The

Structure of the Ubiquinol Oxidase from Escherichia coli and its Ubiquinone

Binding Site, Nat. Struct. Biol. 7, 910-917.

21. Qin, L., Hiser, C., Mulichak, A., Garavito, R. M., and Ferguson-Miller, S.

(2006) Identification of Conserved Lipid/Detergent- Binding Sites in a

High-Resolution Structure of the Membrane Protein Cytochrome c Oxidase,

Proc. Natl. Acad. Sci. U.S.A. 103, 16117-16122.

22. Soulimane, T., Buse, G., Bourenkov, G. P., Bartunik, H. D., Huber, R., and

Than, M. E. (2000) Structure and Mechanism of the Aberrant ba3-cytochrome

c Oxidase from Thermus thermophilus, EMBO J. 19, 1766-1776.

77

23. Hemp, J., Robinson, D. E., Ganesan, K. B., Martinez, T. J., Kelleher, N. L.,

and Gennis, R. B. (2006) Evolutionary Migration of a Post-Translationally

Modified Active-Site Residue in the Proton-Pumping Heme-Copper Oxygen

Reductases, Biochemistry 45, 15405-15410.

24. Buse, G., Soulimane, T., Dewor, M., Meyer, H. E., and Blu g̈gel, M. (1999)

Evidence for a Copper-Coordinated Histidine-Tyrosine Cross-Link in the

Active Site of Cytochrome Oxidase, Protein Sci. 8, 985-990.

25. Rauhamaki, V., Baumann, M., Soliymani, R., Puustinen, A., and Wikstrom, M.

(2006) Identification of a histidine-tyrosine crosslink in the active site of the

cbb3-type cytochrome c oxidase from Rhodobacter sphaeroides, Proc. Natl.

Acad. Sci. U.S.A. 103, 16135-16140.

26. Hemp, J., Christian, C., Barquera, B., Gennis, R. B., and Martinez, T. J. (2005)

Helix Switching of a Key Active-Site Residue in the Cytochrome cbb3

Oxidases, Biochemistry 44, 10766-10775.

27. Hosler, J. P., Ferguson-Miller, S., Calhoun, M. W., Thomas, J. W., Hill, J.,

Lemieux, L., Ma, J., Georgiou, C., Fetter, J., Shapleigh, J., Tecklenburg, M. M.

J., Babcock, G. T., and Gennis, R. B. (1993) Insight into the Active-Site

Structure and Function of Cytochrome Oxidase by Analysis of Site-Directed

Mutants of Bacterial Cytochrome aa3 and Cytochrome bo, J. Bioenerg.

Biomembr. 25, 121-136.

28. Gennis, R. B. (1998) Multiple Proton-conducting Pathways in Cytochrome

Oxidase and a Proposed Role for the Active-Site Tyrosine, Biochim. Biophys.

Acta 1365, 241-248.

29. Gennis, R. B. (2004) Coupled Proton and Electron Transfer Reactions in

Cytochrome Oxidase, Front. Biosci. 9, 581-591.

78

30. Namslauer, A., Pawate, A., Gennis, R. B., and Brzezinski, P. (2003)

Redox-Coupled Proton Translocation in Biological Systems: Proton Shuttling

in Cytochrome c Oxidase, Proc. Natl. Acad. Sci. U.S.A. 100, 15543-15547.

31. Pfitzner, U., Hoffmeier, K., Harrenga, A., Kannt, A., Michel, H., Bamberg, E.,

Richter, O.-M. H., and Ludwig, B. (2000) Tracing the D-Pathway in

Reconstituted Site-Directed Mutants of Cytochrome c Oxidase from

Paracoccus denitrificans, Biochemistry 39, 6756-6762.

32. Branden, M., Sigurdson, H., Namslauer, A., Gennis, R. B., A¬ delroth, P., and

Brzezinski, P. (2001) On the Role of the K-proton Transfer Pathway in

Cytochrome c Oxidase, Proc. Natl. Acad. Sci. U.S.A. 98, 5013-5018.

33. Branden, M., Tomson, F. L., Gennis, R. B., and Brzezinski, P. (2002) The

Entry Point of the K-Proton-Transfer Pathway in Cytochrome c Oxidase,


34. Konstantinov, A. A., Siletsky, S., Mitchell, D., Kaulen, A., and Gennis, R. B.

(1997) The Roles of the Two Proton Input Channels in Cytochrome c Oxidase

from Rhodobacter sphaeroides Probed by the Effects of Site-Directed

Mutations on Time-Resolved Electrogenic Intraprotein Proton Transfer, Proc.

Natl. Acad. Sci. U.S.A. 94, 9085-9090.

35. Yoshikawa, S., Muramoto, K., Shinzawa-Itoh, K., Aoyama, H., Tsukihara, T.,

Ogura, T., Shimokata, K., Katayama, Y., and Shimada, H. (2006) Reaction

Mechanism of Bovine Heart Cytochrome c Oxidase, Biochim. Biophys. Acta

1757, 395-400.

36. Yoshikawa, S. (2003) A Cytochrome c Oxidase Proton Pumping Mechanism

that Excludes the O2 Reduction Site, FEBS Lett. 555, 8-12.

37. Shimokata, K., Katayama, Y., Murayama, H., Suematsu, M., Tsukihara, T.,

79

Muramoto, K., Aoyama, H., Yoshikawa, S., and Shimada, H. (2007) The

Proton Pumping Pathway of Bovine Heart Cytochrome c Oxidase, Proc. Natl.

Acad. Sci. U.S.A. 104, 4200- 4205.

38. Lee, H.-m., Das, T. K., Rousseau, D. L., Mills, D. A., Ferguson-Miller, S., and

Gennis, R. B. (2000) Mutations in the Putative H-Channel in the Cytochrome

c Oxidase from Rhodobacter sphaeroides Show That This Channel Is Not

Important for Proton Conduction but Reveal Modulation of the Properties of

Heme a, Biochemistry 39, 2989-2996.

39. Salje, J., Ludwig, B., and Richter, O. M. (2005) Is a Third Proton-Conducting

Pathway Operative in Bacterial Cytochrome c Oxidase?, Biochem. Soc. Trans.

33, 829-831.

40. Branden, G., Gennis, R. B., and Brzezinski, P. (2006) Transmembrane Proton

Translocation by Cytochrome c Oxidase, Biochim. Biophys. Acta 1757,

1052-1063.

41. Wraight, C. A. (2006) Chance and DesignsProton Transfer in Water, Channels

and Bioenergetic Proteins, Biochim. Biophys. Acta 1757, 886-912.

42. Olsson, M. H. M., Sharma, P. K., and Warshel, A. (2005) Simulating Redox

Coupled Proton Transfer in Cytochrome c Oxidase: Looking for the Proton

Bottleneck, FEBS Lett. 579, 2026-2034.

43. Kato, M., Pisliakov, A. V., and Warshel, A. (2006) The Barrier for Proton

Transport in Aquaporins as a Challenge for Electrostatic Models: The Role of

Protein Relaxation in Mutational Calculations, Proteins 64, 829-844.

44. Arslan, E., Kannt, A., Thony-Meyer, L., and Hennecke, H. (2000) The

Symbiotically Essential cbb(3)-Type Oxidase of Bradyrhizobium japonicum Is

a Proton Pump, FEBS Lett. 470, 7-10.

80

45. Toledo-Cuevas, M., Barquera, B., Gennis, R. B., Wikstrom, M., and

Garcı´a-Horsman, J. A. (1998) The cbb3-Type Cytochrome c Oxidase from

Rhodobacter sphaeroides, a Proton Pumping Heme-Copper Oxidase, Biochim.

Biophys. Acta 1365, 421-434.

46. Sone, N., Tsukita, S., and Sakamoto, J. (1999) Direct Correlationship Between

Proton Translocation and Growth Yield: An Analysis of the Respiratory Chain

of Bacillus stearothermophilus, J. Biosci. Bioeng. 87, 495-499.

47. Sharma, V., Puustinen, A., Wikstrom, M., and Laakkonen, L. (2006) Sequence

Analysis of the cbb3 Oxidases and an Atomic Model for the Rhodobacter

sphaeroides Enzyme, Biochemistry 45, 5754-5765.

48. Altschul, S. F., Gish, W., Miller, W., Myers, E. W., and Lipman, D. J. (1990)

Basic Local Alignment Search Tool, J. Mol. Biol. 215, 403-410.

49. Edgar, R. C. (2004) MUSCLE: Multiple Sequence Alignment with High

Accuracy and High Throughput, Nucleic Acids Res. 32, 1792-1797.

50. Clamp, M., Cuff, J., Searle, S. M., and Barton, G. J. (2004) The Jalview Java

Alignment Editor, Bioinformatics 20, 426-427.

51. Milpetz, F., Argos, P., and Persson, B. (1995) TMAP: A New Email and

WWW Service for Membrane-Protein Structural Predictions, Trends Biochem.

Sci. 20, 204-205.

52. Guex, N., and Peitsch, M. C. (1997) SWISS-MODEL and the

Swiss-PdbViewer: An Environment for Comparative Protein Modeling,

Electrophoresis 18, 2714-2723.

53. Zhang, L., and Hermans, J. (1996) Hydrophilicity of Cavities in Proteins,

Proteins 24, 433-438.

54. Phillips, J. C., Braun, R., Wang, W., Gumbart, J., Tajkhorshid, E., Villa, E.,

81

Chipot, C., Skeel, R. D., Kale, L., and Schulten, K. (2005) Scalable Molecular

Dynamics with NAMD, J. Comput. Chem. 26, 1781-1802.

55. Foloppe, N., and MacKerell, A. D., Jr. (2000) All-Atom Empirical Force Field

for Nucleic Acids: I. Parameter Optimization Based on Small Molecule and

Condensed Phase Macromolecular Target Data, J. Comput. Chem. 21, 86-104.

56. Oh, J. I. (2006) Effect of Mutations of Five Conserved Histidine Residues in

the Catalytic Subunit of the cbb3 Cytochrome c Oxidase on Its Function, J.

Microbiol. 44, 284-292.

57. Thomas, P. E., Ryan, D., and Leven, W. (1976) An Improved Straining

Procedure for the Detection of the Peroxidase Activity of Cytochrome P-450

on Sodium Dodecyl Sulfate Polyacrylamide Gels, Anal. Biochem. 75,

168-176.

58. DeSantis, T. Z., Hugenholtz, P., Larsen, N., Rojas, M., Brodie, E. L., Keller, K.,

Huber, T., Dalevi, D., Hu, P., and Andersen, G. L. (2006) Greengenes, a

Chimera-Checked 16S rRNA Gene Database and Workbench Compatible with

ARB, Appl. EnViron. Microbiol. 72, 5069-5072.

59. Thorndycroft, F. H., Butland, G., Richardson, D. J., and Watmough, N. J.

(2007) A New Assay for Nitric Oxide Reductase Reveals Two Conserved

Glutamate Residues Form the Entrance to a Proton-Conducting Channel in the

Bacterial Enzyme, Biochem. J. 401, 111-119.

60. Hendriks, J., Oubrie, A., Castresana, J., Urbani, A., Gemeinhardt, S., and

Saraste, M. (2000) Nitric Oxide Reductases in Bacteria, Biochim. Biophys.

Acta 1459, 266-273.

61. Pawate, A. S., J., M., Namslauer, A., Mills, D. A., Brzezinski, P.,

Ferguson-Miller, S., and Gennis, R. B. (2002) A Mutation in Subunit I of

82

Cytochrome Oxidase from Rhodobacter sphaeroides Results in an Increase in

Steady-State Activity but Completely Eliminates Proton Pumping,


62. Siletsky, S. A., Pawate, A. S., Weiss, K., Gennis, R. B., and Konstantinov, A. A.

(2004) Transmembrane Charge Separation During the Ferryl-Oxo Oxidized

Transition in a Nonpumping Mutant of Cytochrome c Oxidase, J. Biol. Chem.

279, 52558-52565.

63. Han, D., Morgan, J. E., and Gennis, R. B. (2005) G204D, a Mutation That

Blocks the Proton-Conducting D-Channel of the aa3-Type Cytochrome c

Oxidase from Rhodobacter sphaeroides, Biochemistry 44, 12767-12774.

64. Pomes, R., and Roux, B. (1996) Structure and Dynamics of a Proton Wire: A

Theoretical Study of H+ Translocation Along the Single-File Water Chain in

the Gramicidin A Channel, Biophys. J. 71, 19-39.

65. Wraight, C. A. (2005) Intraprotein Proton TransfersConcepts and Realities

from the Bacterial Photosynthetic Reaction Center, in Biophysical and

Structual Aspects of Bioenergetics (Wı¨kstrom, M., Ed.) pp 273-312, Royal

Society of Chemistry, Cambridge.

66. Ferguson-Miller, S., and Babcock, G. T. (1996) Heme/Copper Terminal

Oxidases, Chem. ReV. 7, 2889-2907.

67. Adelroth, P., Paddock, M. L., Sagle, L. B., Feher, G., and Okamura, M. Y.

(2000) Identification of the Proton Pathway in Bacterial Reaction Centers:

Both Protons Associated with Reduction of QB to QBH2 Share a Common

Entry Point, Proc. Natl. Acad. Sci. U.S.A. 97, 13086-13091.

68. Adelroth, P., Paddock, M. L., Tehrani, A., Beatty, J. T., Feher, G., and Okamura,

M. Y. (2001) Identification of the Proton Pathway in Bacterial Reaction

83

Centers: Decrease of Proton Transfer Rate by Mutation of Surface Histidines

at H126 and H128 and Chemical Rescue by Imidazole Identifies the Initial

Proton Donors, Biochemistry 40, 14538-14546.

69. Miller, M. J., Oldenburg, M., and Fillingame, R. H. (1990) The Essential

Carboxyl Group in Subunit c of the F1F0 ATP Synthase Can Be Moved and

H+-translocating Function Retained, Proc. Natl. Acad. Sci. U.S.A. 87,

4900-4904.

70. Fillingame, R. H., Angevine, C. M., and Dmitriev, O. Y. (2003) Mechanics of

Coupling Proton Movements to C-Ring Rotation in ATP Synthase, FEBS Lett.

555, 29-34.

71. Crofts, A. R., Hong, S., Ugulava, N., Barquera, B., Gennis, R.,

Guergova-Kuras, M., and Berry, E. A. (1999) Pathways for Proton Release

During Ubihydroquinone Oxidation by the bc1 complex, Proc. Natl. Acad. Sci.

U.S.A. 96, 10021-10026.

72. Izrailev, S., Crofts, A. R., Berry, E. A., and Schulten, K. (1999) Steered

Molecular Dynamics Simulation of the Rieske Subunit Motion in the

Cytochrome bc(1) Complex, Biophys. J. 77, 1753-1768.

73. Huang, L. S., Cobessi, D., Tung, E. Y., and Berry, E. A. (2005) Binding of the

Respiratory Chain Inhibitor Antimycin to the Mitochondrial bc1 Complex: A

New Crystal Structure Reveals an Altered Intramolecular Hydrogen-Bonding

Pattern, J. Mol. Biol. 351, 573-597.

74. Hunte, C., Koepke, J., Lange, C., Rossmanith, T., and Michel, H. (2000)

Structure at 2.3 Å Resolution of the Cytochrome bc(1) Complex from the

Yeast Saccharomyces cereVisiae Co-crystallized with an Antibody Fv

Fragment, Structure 8, 669-684.

84

75. Lanyi, J. K. (2006) Proton Transfers in the Bacteriorhodopsin Photocycle,

Biochim. Biophys. Acta 1757, 1012-1018.

76. Horsefield, R., Yankovskaya, V., Sexton, G., Whittingham, W., Shiomi, K.,

Omura, S., Byrne, B., Cecchini, G., and Iwata, S. (2006) Structural and

Computational Analysis of the Quinone-Binding Site of Complex II

(Succinate-Ubiquinone Oxidoreductase): A Mechanism of Electron Transfer

and Proton Conduction during Ubiquinone Reduction, J. Biol. Chem. 281,

7309-7316.

77. Madej, M. G., Nasiri, H. R., Hilgendorff, N. S., Schwalbe, H., and Lancaster,

C. R. (2006) Evidence for Transmembrane Proton Transfer in a

Dihaem-Containing Membrane Protein Complex, EMBO J. 25, 4963-4970.

78. Tomson, F. L., Morgan, J. E., Gu, G., Barquera, B., Vygodina, T. V., and

Gennis, R. B. (2003) Substitutions for Glutamate 101 in Subunit II of

Cytochrome c Oxidase from Rhodobacter sphaeroides Result in Blocking the

Proton-Conducting K-Channel, Biochemistry 42, 1711-1717.

79. Zufferey, R., Arsian, E., Thony-Meyer, L., and Hennecke, H. (1998) How

Replacements of the 12 Conserved Histidines of Subunit I Affect Assembly,

Cofactor Binding, and Enzymatic Activity of the Bradyrhizobiuim japonicum

cbb3-Type Oxidases, J. Biol. Chem. 273, 6452-6459.

80. Ozturk, M., and Mandaci, S. (2006) Two Conserved Non-canonical Histidines

Are Essential for Activity of the cbb(3)-Type Oxidase in Rhodobacter

capsulatus, Mol. Biol. Rep. (in press).

81. Mitchell, D. M., Fetter, J. R., Mills, D. A., Adelroth, P., Pressler, M. A., Kim,

Y., Aasa, R., Brzezinski, P., Malmstrom, B. G., Alben, J. O., Babcock, G. T.,

Ferguson-Miller, S., and Gennis, R. B. (1996) Site-Directed Mutagenesis of

85

Residues Lining a Putative Proton Transfer Pathway in Cytochrome c Oxidase

from Rhodobacter sphaeroides, Biochemistry 35, 13089-13093.

82. de Vries, S., and Schroder, I. (2002) Comparison between the Nitric Oxide

Reductase Family and Its Aerobic Relatives, the Cytochrome Oxidases,

Biochem. Soc. Trans. 30, 662-667.

83. Belevich, I., Tuukkanen, A., Wikstrom, M., and Verkhovsky, M. I. (2006)

Proton-Coupled Electron Equilibrium in Soluble and Membrane-Bound

Cytochrome c Oxidase from Paracoccus denitrificans, Biochemistry 45,

4000-4006.

84. Wikstrom, M., Verkhovsky, M. I., and Hummer, G. (2003) Water-Gated

Mechanism of Proton Translocation by Cytochrome c Oxidase, Biochim.

Biophys. Acta 1604, 61-65.

85. Morgan, J. E., Verkhovsky, M. I., and Wikstrom, M. (1994) The Histidine

Cycle: A New Model for Proton Translocation in the Respiratory

Heme-Copper Oxidases, J. Bioenerg. Biomembr. 26, 599-608.

86. Michel, H. (1999) Cytochrome c Oxidase: Catalytic Cycle and Mechanisms of

Proton PumpingsA Discussion, Biochemistry 38, 15129-15140.

86

CHAPTER 4: ROLE OF CALCIUM BINDING SITES IN CBB3–TYPE CYTOCHROME C OXIDASE

4.1 Abstract

C-family (cbb3–type oxidase) cytochrome c oxidase contains six redox active

metal centers including two heme b, three heme c and one CuB. Scarce information is

known about the nonredox metal ions in this type of oxidase. We analyzed the metal

composition of cbb3–type oxidases from Rhodobacter sphaeroides and Vibrio

Cholerae through ICP-OES assay and found out other than the redox active metals

only two calcium ions existed as the redox inactive metal groups. The very recent

crystal structure from Pseudomonas stutzeri (personal communication with Dr.

Hartmut Michel) identified the binding site of one Ca2+

but not the other. This

identified Ca2+

connects subunit CcoN and CcoO and bridges heme b and heme b3

through the propionate groups. Site-directed mutagenesis studies of the binding

residues E180 (subunit CcoN) and S105 (subunit CcoO, R. sphaeroides numbering)

indicate that they are essential for the activity of the enzyme and recruitment of both

Calcium ions and CuB. Mutagenesis study of another conserved nearby residue E183

suggests that this acidic residue is also required for the correct incorporation of

calcium and copper ions and necessary for the fully functional enzyme.

4.2 Introduction

In the respiratory chain of mitochondria and bacteria cytochrome c oxidases

(COXs) function as the terminal enzymes to catalyze the reduction of molecular

oxygen to water coupling with the proton translocation from the cytoplasmic side to

the periplasmic side of the membrane [1-3]. The proton gradient formed provides the

primary energy source for ATP synthesis. These enzymes uniformly contain a

87

heme-copper binuclear center which is consisting of a high-spin heme (one histidine

ligand) and a nearby copper (three histidine ligands) where the reduction of oxygen

could happen. The major catalytic subunit also contains another low-spin heme which

is coordinated by two fully conserved histidines. The other common feature they

share is that a well conserved tyrosine close to the active site is covalently linked to

one of the histidine ligands of CuB.

COXs could be classified into three major families (A, B and C type) based on

the structural analysis, sequence alignments and proton pumping mechanisms [4, 5].

A-type family is the most abundantly found and widely characterized COX which is

represented by aa3 cytochrome c oxidase. It has another redox center CuA which is

located in the subunit II and helps to deliver the electrons to binuclear center. Apart

from the redox centers, X-ray crystal structures have revealed that aa3-type oxidase

contains several other metal centers like Zn2+

, Mg2+

and cations that are not redox

active [6-11]. Mutagenesis studies have confirmed two different proton translocation

channels (D and K) which move the protons to active site for formation of water or

simply pump protons across the membrane [12,13]. B-type family is represented by

ba3–type cytochrome c oxidase which carries only one proton input channel [14].

Crystal structure of ba3–type oxidase revealed no additional metals other than copper

and irons [15].

C-type family which is represented by cbb3–type cytochrome c oxidase is the

second largest group (24%) and is mainly found in bacteria [4]. It has the ability to

maintain its catalytic activity under low-oxygen conditions and in the mean time

pump protons across the membrane as it has high affinity to oxygen [16]. Sequence

alignment and mutagenesis study have shown that cbb3–type oxidase has only one

proton input channel (K-channel) [17] like B-type oxidase and pump protons less

88

efficiently than A-type oxidase [18]. cbb3 oxidase is encoded by four clustered genes

ccoNOQP which are positively regulated by ccoGHIS [19]. Subunit CcoN contains

the catalytic center consisting of a high-spin heme b3 and a nearby copper atom (CuB).

A low-spin heme b is located close to the active site to deliver the electrons. Unlike

the A and B-type oxidase, cbb3 oxidase has no CuA in the subunit II. Instead it harbors

one and two c-type hemes in the transmembrane subunits CcoO and CcoP

respectively [20, 21] which could serve as the electron acceptors from bc1 complex.

The smallest subunit CcoQ has no associated cofactor and the function is not very

clear so far. Except subunit CcoN the rest three subunits share no homology with

other types of cytochrome c oxidase.

Crystal structures of A and B-type oxidases have provided clear images about

the binding sites of the redox and non-redox metal groups. There is significant

difference between these cytochrome c oxidases regarding the content of calcium ion.

In mammalian mitochondria, aa3–type oxidase has the common sites which could

bind Ca2+

or Na+ reversibly [8, 22]. However, in bacteria strains like Paracoccus

denitrificans and R. sphaeroides, the aa3–type oxidases contain the calcium ions that

bind strongly with the conserved residues [10, 11, 23-25]. Although the identity of

this metal site cannot be definitely confirmed by the crystal structure, the assignment

of Na+ is preferred in bovine heart mitochondria whereas Ca

2+ is preferred in bacteria

P. denitrificans and R. sphaeroides. This cation site is quite similarly located in the

periplasmic side membrane border of subunit I in aa3–type oxidase from bovine heart

mitochondria or bacteria strains. But the red shift in heme a absorption spectrum upon

adding Ca2+

is exhibited only in bovine COX not in P. denitrificans or R. sphaeroides

[26, 27]. The functional and structural role of this metal site is still not clear as there

are no significant effects on electron transfer or proton translocation caused by Ca2+

or

89

Na+. This cation binding site is missing in the B-type oxidase from Thermus

thermophilus [15].

Due to the lack of structure of cbb3–type cytochrome c oxidase, there has been

no report about the non-redox metal ions. Until recently Dr. Hartmut Michel just

solved the first crystal structure of cbb3–type oxidase from P. stutzeri (manuscript in

preparation). The calcium binding sites were identified and this forms the basis of our

mutagenesis studies. Through sequence alignment we identified the conserved Ca2+

binding residues in R. sphaeroides and V. Cholerae. Then site-directed mutagenesis

was applied systematically to generate series of mutants at the Ca2+

binding sites.

These studies suggest that the Ca2+

binding residues are not only critical for the

functionality but also important for the stability of cbb3–type oxidase.


Mutagenesis of the cbb3-Type Oxidase from R. sphaeroides. Site-directed mutagenesis

and cloning were performed as previously reported using the expression plasmid

pUI2803NHIS which contains six histidine codons at the C-terminus of subunit ccoN

[28]. Point mutations of subunit ccoN and ccoO were made by quick-change PCR in

plasmid pUC19 using Quick-change mutagenesis kit from Stratagen and then

subcloned into expression plasmid pUI2803NHIS. The quick change PCR primers

used for mutagenesis were synthesized by Integrated DNA Technologies (IDT). The

resulting mutant expression plasmids were transformed into E. coli S-17-1 by

electroporation and then transferred to R. sphaeroides 2.4.1 ccoNOQP deletion strain

by conjugation. Sequence verification of the mutagenesis products was carried out at

the Biotechnology Center at the University of Illinois, Urbana-Champaign.

Mutagenesis of the cbb3-Type Oxidase from V. cholerae. Site-directed mutagenesis

90

was performed using Stratagene QuickChange kits as previously reported [29]. The

quick change PCR primers used for mutagenesis were synthesized by Integrated DNA

Technologies (IDT). Sequence verification of the mutagenesis reactions was

performed at the Biotechnology Center at the University of Illinois,

Urbana-Champaign.

Purification of the His-tagged cbb3-Type Oxidases. The wild-type and mutant cbb3

proteins from R. sphaeroides and V. cholerae with polyhistidine tags were

overexpressed and purified as previously described [28, 29]. R. sphaeroides was

grown semi-aerobically at 30°C in Sistrom media with 2 µg/mL tetracycline (Sigma).

V. cholerae cells were grown aerobically at 37°C in LB media (USB Corp.) with 100

µg/mL ampicillin (Fisher Biotech) and 100 µg/mL streptomycin (Sigma). cbb3

oxidase overexpression in V. cholerae was induced with 0.2% L-(+)- arabinose

(Sigma). The cells were collected by centrifugation at 7000 rpm for 15 min when their

growth reached early stationary phase and re-suspended in 20mM Tris buffer at pH

8.0 with 10mM MgCl2, 100mM NaCl, DNaseI and protease-inhibitor cocktail. Then

the cells were lysed by microfluidizer (Watts Fluidair, Inc.) and centrifuged at 40,000

rpm for 4 hours to collect the membranes. Afterwards the membranes were

solubilized with 1% dodecyl-D-maltoside (Anatrace) for 2 hours. Nonsolubilized

membranes were removed by centrifuging at 40000 rpm for 30 min. The solubilized

protein was mixed with Ni2+

-NTA resin (Qiagen, CA) in the presence of 5mM

imidazole for 2 hours and then purified through gravity column (Bio-rad). The

column with resin was washed with 10 column volumes of Tris buffer with 20mM

imidazole and the protein with red color was eluted using 200mM imidazole. The

purified protein was dialyzed with 20mM Tris buffer at pH 7.5 with 30mM KCl, 1mM

EDTA and 0.05% DDM and concentrated with Amicon concentrator with 100 kD

91

cut-off (Millipore).

Heme Analysis of Proteins. As the heme c is covalently attached to subunit CcoO and

CcoP, heme staining was used to identify these two subunits [30]. GeneMate Express


The gels were then incubated in dark by 15ml 6.3 mM 3,3’,5,5’-tetramethylbenzidine

(TMBZ from Sigma) and 35ml 0.25 M sodium acetate, pH 5.0, for 1 h. The gels were

then stained for heme c by adding H2O2 to 30 mM. After 30 minutes incubation the

gels were washed by 3 parts isopropanol and 7 parts 0.25 M sodium acetate, pH 5.0.

UV/Vis Spectroscopic Analysis. Shimadzu UV/Vis-2101PC spectrophotometer was

used to obtain the spectra of wild-type and mutant enzymes. The concentrated protein

samples were diluted with 20mM Tris buffer and 0.05% DDM at pH 7.5. The

enzymes were oxidized by air and reduced with sodium dithionite (Sigma). Spectra

were measured from 300 to 800 nm and analyzed using Origin.

Pyridine Hemochrome Spectra Assay. The concentrations of heme b and heme c were

determined as previously described [31]. 0.5ml of a stock solution containing 200mM

NaOH and 40% (by volume) pyridine and 3µl of 0.1M K3Fe(CN)6 were placed in a

1ml cuvette. A 0.5ml aliquot of the protein sample (~5mM) was added with thorough

mixing and oxidized spectrum was recorded within 1 minute. Solid sodium dithionite

(2-5mg) was then added and several successive spectra of the reduced pyridine

hemochromes were recorded. The absorbance differences at the selected wavelengths

were multiplied as a vector by the inverse of the matrix of extinction coefficients at

these wavelengths to obtain the concentration of heme b and heme c.

Steady-state Activity Measurements. A YSI model 53 oxygen monitor was used to

polarographically measure steady-state oxidase activity at 25°C. 1.8 mL of 50 mM

sodium phosphate and 100 mM NaCl buffer at pH 6.5 containing 0.05% DDM was

92

mixed with 10µL 1 M ascorbate, pH 7.4, and 20µL of 0.1 M TMPD in the sample

chamber. Horse heart cytochrome c (Sigma) was added as the substrate to a final

concentration of 40 µM. The reaction was initiated by adding 10 µL of 1 µM enzyme,

and oxygen consumption rate was monitored by the oxygen meter. The enzyme

turnover (electron/second/enzyme) was calculated based on the relative slope of the

oxygen-consumption traces.

Metal Content Analysis. Metal content of the protein was determined by inductively

coupled plasma optical emission spectroscopy (ICP-OES) using a Spectro Genesis

spectrometer as previously described [32]. Protein samples were incubated with 1 mM

EDTA and 100 µM EGTA prior to buffer exchange and analysis. Metal concentrations

were calculated from regression lines of element standards run as samples rather than

the regression lines produced by the Spectro software to avoid a 10% underestimation

of element concentrations when the element concentration in the sample is low.

4.4 Results

The cbb3–type oxidases from R. sphaeroides and V. Cholerae were analyzed

for their metal contents using ICP-OES. Both of the wild-type enzymes showed quite

similar pattern and no Mg, Mn, Mo or Zn could be detected. Other than the redox

active metal centers, Cu and Fe, only Ca ion was found to be the non-redox active

metal. And the stoichiometry of Ca/Cu was shown to be around 2 for both of the

wild-type enzymes from R. sphaeroides and V. Cholerae (Table 1). Due to the

impurities it’s not accurate to estimate the concentration of enzyme based on the

sulfur content. Instead copper was used as the calibration. As there is one copper in

one oxidase monomer, two calcium ions are expected in one enzyme. Both enzymes

were treated with buffer exchange in the presence of 1 mM EDTA. Non-specific

93

bound metals should be removed by the chelating process. Therefore these two Ca

irons should bind firmly with the protein complex.

The crystal structure of cbb3–type oxidase from P. stutzeri only clearly

presented one calcium ion with identities of two binding ligands. One is Glu122

located in subunit CcoN and the other one Ser102 is from subunit CcoO. Sequence

alignment was used to identify the correspondingly conserved residues in cbb3–type

oxidases from R. sphaeroides and V. Cholerae (Fig. 1). Glu180 and Ser105 from

subunit CcoN and CcoO respectively coordinate one calcium ion in cbb3–type

oxidases from R. sphaeroides. Similarly Glu126 and Ser107 from subunit CcoN and

CcoO respectively ligate one calcium ion in cbb3–type oxidases from V. Cholerae.

The glutamate residue is fully conserved among all the cbb3–type oxidases and the

serine residue from CcoO is also quite highly conserved (>91%).

Mutagenesis studies were carried out as listed in Table 2. Since another fully

conserved glutamate residue is quite closed to the calcium binding glutamate (2 amino

acids apart) it was also selected for the study. Those glutamates were mutated to

glutamine, aspartate or glycine to test the different effects of side chain changes. The

serine site from subunit CcoO was substituted with threonine, alanine or glycine to

introduce possible structural perturbation. All the mutant proteins were expressed with

His-tag and purified to homogeneity by nickel-affinity columns. The purified enzymes

were assayed by SDS-PAGE gels for correct subunits and analyzed by pyridine

hemochrome spectra for heme composition (Table 2). Heme c was stained in the

SDS-PAGE gel to confirm the location of subunit CcoO and CcoP (Fig. 2). All the

purified mutant proteins exhibited electron transfer disability. The C-type oxidase

mutants S105T, S105A and S105G from R. sphaeroides all showed assembly defects

and no recombinant protein complex could be detected or purified. This indicates that

94

this serine residue from subunit CcoO plays a very important role for maintaining the

correct structure. The glycine substitution of either of the glutamates in subunit CcoN

from R. sphaeroides lead to instability of the enzyme and very little protein could be

obtained. And the heme c to heme b ratios were substantially altered for E180G (2:1)

and E183G (4:1). E180D, E180Q, E183D and E183Q showed quite similar subunits

and heme ratio pattern like wild-type cbb3–type oxidase, but their oxygen turn over

activities were abolished. Whereas in V. Cholerae all the equivalent glutamate mutant

proteins E126D, E126Q, E126G, E129D, E129Q and E129G were purified to certain

amount for characterization but with much less yield than wild-type oxidase. These

mutants contained the correct subunits and their heme ratios were all around 3:2 like

the wild-type (Table 2). But all the mutant proteins were virtually inactive.

Elements analysis by ICP-OES showed that any mutation of either of the

conserved glutamates completely eliminated CuB in cbb3–type oxidases from R.

sphaeroides or V. Cholerae. From the crystal structure only one glutamate was

identified to be the ligand of one calcium ion and it is corresponding to E180 (R.

sphaeroides) or E126 (V. Cholerae). But both of the Ca ions were lost in any of the

single mutations of this glutamate which suggested that this residue could be

associated with both of the Ca ions somehow. It came as a surprise that the second

glutamate E183 (R. sphaeroides) or E126 (V. Cholerae) was also intimately connected

to these two calcium ions because the mutation of this Glutamate to Glutamine caused

significant loss of Ca and Cu. These findings were consistent with the reduced spectra

analysis results which indicated that heme b in these mutant cbb3 proteins could not

be reduced by dithionite because the diagnostic shoulder peak at 561nm was missing

in the reduced-oxidized spectra (Fig. 3). As the copper was removed electrons could

not be delivered to heme b or subsequently to binuclear center. It should be noted that

95

the mutatant E183Q retained all the calciums and copper like in the wildtype enzyme.

And correspondently this mutant has relatively higher activity than all the other

Glutamate mutants (Table 2).

4.5 Discussion

Two calcium ions found as the non-redox metal

Similar to other heme-copper oxidases, C-type oxidase contains heme irons and

copper in the active site. The finding that this type of oxidase has two calcium ions

and no other redox inactive metals suggests it is quite distinctive from all the other

cytochrome c oxidases in the respect of structure and functionality. The ion Mg2+

or

Mn2+

which holds together subunit I and II in A-type oxidase does not exist in C-type

oxidase. One calcium ion instead substitutes the role of associating the first two

subunits in cbb3–type oxidase. The prolonged dialysis with EDTA did not remove

Ca2+

from the cbb3–type oxidases. This fact indicates that the calcium ions might have

quite high affinity with the binding sites in cbb3–type oxidases which share some

similarity with other aa3–type oxidases from bacteria [24, 25]. There is no spectrum

shift of heme peaks caused by adding of Ca2+

which was exhibited in bovine aa3–type

oxidase [33, 34]. Accordingly there is no competition of Na+ or H

+ with Ca

2+ in

C-type oxidase, which confirms the peculiar role of Ca2+

in C-type oxidase.

Identity of the Ca2+

-binding sites

It has been shown that the Ca2+

binding site for aa3–type oxidase from bovine

heart or bacteria is formed by the residues at the periplasmic side in subunit I and is

closer to heme a than to heme a3 [8, 10, 11]. The structures of the bacterial and

mitochondrial Ca2+

binding sites are quite similar even with little differences. The

calcium ion is coordinated by five bonds which are partially conserved in bacterial

and mammalian enzymes.

96

The new crystal structure of cbb3–type oxidase from P. stutzeri clearly shows

one Ca2+

binding site leaving the other one in ambiguity. One of the calcium ions is

coordinated by Glu122 from subunit CcoN, Ser102 from subunit CcoO and two

propionate groups from hemes b and b3. These two residues are quite conserved in the

C-type oxidases. The structure of this calcium binding site is quite different with the

one from A-type oxidases, but resembles the calcium binding site from qNOR nitric

oxide reductase (new crystal structure by Dr. Yoshitsugu Shiro). This calcium ion

from qNOR or cbb3-type oxidase connects the low-spin heme b and high-spin heme

b3 in a similar way and shares the conserved Glutamate binding ligand. This is

another piece of evidence that C-type oxidase and nitric oxide reductase are closely

related with each other in the evolutionary aspect in addition to the conserved

sequence features [17].

Although the other Ca2+

binding site has not been clearly identified, our

mutagenesis studies of the conserved double Glutamate residues (E180 and E183, R.

sphaeroides numbering) suggest that both of them are closely associated with these

two calcium ions because any mutations of E180 eliminate all the calcium and E183D

significantly reduces the content of calcium (Table 1). This is also observed in V.

Cholerae. The fact that one calcium ions are intact in mutant E183Q suggests that the

propionate group is required to attach both of the two calcium ions. No assembled

S105 mutant (subunit CcoO, R. sphaeroides numbering) could be purified, therefore

we have no way to characterize it further. But it is reasonable to propose that this

residue plays a crucial role in connecting subunit CcoN and CcoO and is required for

the whole protein complex assembly.

Roles of Ca2+

-binding sites

There has been no report about the significant effect of Ca2+

on the activity or

97

proton pumping capacity of A-type oxidases from bacteria or mitochondria and the

functional significance of the Ca2+

binding site is not clear so far. But here the

mutagenesis study of the Ca2+

binding sites in C-type oxidases strongly indicates that

the calcium ions are vital for the activity of the enzyme. The elimination of any Ca2+

is associated with the defect of electron transfer. Except E183Q all the other Glu180

and Glu183 mutants knock out the Ca2+

and CuB in the same time which explains why

the electrons could not be delivered to the heme b3 in the binuclear center (Table 1). It

is not clear how these three metal groups are associated each other. One possible

explanation is that this pair of glutamate residues mediates the calcium ions and CuB

directly or indirectly through water molecules which is not clearly visible in the

crystal structure. It comes as a surprise that substantial loss of activity is also observed

in mutant E183Q where the copper is intact and two Ca2+

ions are present. We

propose that the disturbance of Ca2+

and copper environment might change

substantially the redox potentials of heme b groups causing the electron transfer

failure.

It has been proposed that the conserved glutamate residues in cNOR form a

proton transfer pathway from periplasm to the active site [35, 36]. Similarly the

equivalent glutamates in cbb3–type oxidase were proposed to be the proton exit

pathway to periplasm [17]. But the new qNOR structure shows that there is no

obvious hydrogen bonding network connecting the active site to the periplasm

through these two glutamates. And the new crystal structure of cbb3–type oxidase also

confirms that the first glutamate serves as a ligand of one calcium ion. Our

experimental data here explicitly indicate that these two glutamates are functionally

important for the C-type oxidases and structurally coordinate two calcium ions and

copper. In A-type oxidase the non-redox-active metal site containing Mg2+

/Mn2+

98

sitting above the binuclear center and connecting subunit I and II was proposed to be

involved in the water exit pathway [37]. One of the calcium ions in C-type oxidase

resembles the Mg2+

/Mn2+

site structurally in the way that it associates subunit CcoN

and CcoO and bridges heme b and heme b3. Therefore it is reasonable to deduce that

this Ca2+

site might be part of the water exit channel in C-type oxidase. This proposal

should be examined further by other biophysical methods including the structure

analysis of these mutants.

99


Table 4.1. ICP-OES analysis of cbb3 oxidases

CcO Ca

1

(317.9)

Cu

(327.4)

Fe

(238.2)

Zn

(213.9) Ca/Cu

R.s. WT

cbb3 3.09 1.35 13.72 0 2.29

R. s.

E183Q 3.72 1.61 13.90 0 2.31

R. s.

E183D 0.55

2 Trace 20.42 0 NA

R. s.

E180Q 0 Trace 14.29 0 NA

R. s.

E180D 0 0 7.72 0 NA

V. c. WT

cbb3 4.26 2.21 17.15 Trace 1.93

1. Micromolar; (spectral line in nm) No Mg, Mn or Mo was present in any of the samples.

2. Low concentration; probably within 5% but up to 20% error seems possible

100

Table 4.2. Characteristics of mutant cbb3 oxidases


Location Activity(%)a Assembly

b heme

c/b

Activity(%)a Assembly

b heme

c/b

WT 100 + 1.5 WT 100 + 1.5

CcoN E180D <1 + 1.7 E126D 1 + 1.6

E180Q 1 + 1.6 E126Q <1 + 1.8

E180G <1 - 2 E126G 1 + 1.2

E183D 1 + 1.6 E129D 6 + 1.2

E183Q 7 + 1.5 E129Q 17 + 1.8

E183G 1 - 4 E129G 4 + 1.5

E129A 5 + 1.3

CcoO S105T <1 - NA

S105A <1 - NA

S105G <1 - NA

a Steady state oxidase turnover number measured as described in the text. The activity

of the wild-type cbb3 from R. sphaeroides is about 800 e-/sec/enzyme. And the

activity of the wild-type cbb3 from V. cholerae is about 500 e-/sec/enzyme. Ascorbate

and TMPD were used as the reductants together with horse heart cytochrome c as the

substrate.

b The assembly determination was based on the spectroscopy and SDS-PAGE gel

analysis of the purified proteins.

101

Figure 4.1

LGFTSSKEYAELEWPID

LGATQSKEYAEPEWYLD

LGYTSGKEYAELEWPID

P. stutzeri

R. sphaeroides

V. cholerae

115

173

119

131

189

135

HPFLWGSKRTGPDLARVG

HPFQWGSKRTGPDLARVG

HPFLWGSKRTGPDLARVG

96

99

101

113

116

118

CcoN CcoO

Fig. 4.1. Identities of the calcium binding site of cbb3–type oxidases. The conserved

residues in subunit CcoN and CcoO from different organisms are shown in red. The

Ca2+

binding residues are marked by Δ in blue.

102

Figure 4.2

75 kDa -

25 kDa -

20 kDa -

50 kDa -

37 kDa -

100 kDa -

150 kDa -

CcoN

CcoPCcoO

CcoPCcoO

M 1 2 3 M 1 2 3

A B

4 5 4 5

Fig. 4.2. (A) SDS-PAGE pattern of the purified R. sphaeroides cbb3–type oxidases


stained with Coomassie Brilliant Blue R-250. Subunits CcoN, CcoO, CcoP were

indicated on the right. Lane M stands for Precision Plus Protein Dual Color Standard

marker from Bio-rad. Lane 1 (wild type), lane 2 (E180D), lane 3 (E180Q), lane 4

(E183D), lane 5 (E183Q). (B) The same PAGE gel as (A) was stained with TMBZ to

detect Heme c.

103

Figure 4.3

Fig. 4.3. Difference spectra of R. sphaeroides cbb3–type oxidases. The enzymes were

oxidized naturally by air and reduced by sodium dithionite. The 551 peak is the

indication of cytochrome c and 561 peak is the indication of cytochrome b.

104

4.7 References

[1] Richter, O. M., and Ludwig, B. (2009) Electron transfer and energy transduction

in the terminal part of the respiratory chain — Lessons from bacterial model systems,

Biochim Biophys Acta. 1787, 626-34.

[2] Brzezinski, P., and Gennis, R. B. (2008) Cytochrome c oxidase: Exciting progress

and remaining mysteries, J Bionenerg Biomembr 40, 521–531.

[3] Branden, G., Gennis, R. B., and Brzezinski, P. (2006) Transmembrane proton

translocation by cytochrome c oxidase, Biochim Biophys Acta 1757, 1052–1063.

[4] Hemp, J., and Gennis, R. B. (2008) Diversity of the heme-copper superfamily in

archaea: insights from genomics and structural modeling, Results Probl. Cell. Differ.

45, 1–31.

[5] Pereira, M. M., Santana, M., and Teixeira, M. (2001) A novel scenario for the

evolution of haem-copper oxygen reductases, Biochim Biophys Acta 1505, 185–208.

[6] Tsukihara, T., Aoyama, H., Yamashita, E., Tomizaki, T., Yamaguchi, H.,

Shinzawa-Itoh, K., Nakashima, T., Yaono, R., and Yoshikawa, S. (1995) Structures of

metal sites of oxidized bovine heart cytochrome c oxidase at 2.8 Å, Science 269,

1069-1074.

[7] Tsukihara, T., Aoyama, H., Yamashita, E., Takashi, T., Yamaguichi, H.,

Shinzawa-Itoh, K., Nakashima, R., Yaono, R., and Yoshikawa, S. (1996) The whole

structure of the 13-subunit oxidized cytochrome c oxidase at 2.8 Å, Science 272,

1136-1144.

[8] Yoshikawa, S., Shinzawa-Itoh, K., Nakashima, R., Yaono, R., Inoue, N., Yao, M.,

Fei, M. J., Libeu, C. P., Mizushima, T., Yamaguchi, H., Tomizaki, T., and Tsukihara, T.

(1998) Redoxcoupled crystal structural changes in bovine heart cytochrome c oxidase,

Science 280, 1723-1729.

105

[9] Iwata, S., Ostermeier, C., Ludwig, B., and Michel, H. (1995) Structure at 2.8 Å

resolution of cytochrome c oxidase from Paracoccus denitrificans, Nature 376,

660-669.

[10] Ostermeier, C., Harrenga, A., Ermler, U., and Michel, H. (1997) Structure at 2.7

Å resolution of the Paracoccus denitrificans two subunit cytochrome c oxidase

complexed with an antibody fv fragment, Proc. Natl. Acad. Sci. U.S.A. 94,

10547-10553.

[11] Svensson-Ek, M., Abramson, J., Larsson, G., Tornroth, S., Brzezinski, P., and

Iwata, S. (2002) The X-ray crystal structures of wild-type and EQ(I-286) mutant

cytochrome c oxidases from Rhodobacter sphaeroides, J. Mol. Biol. 321, 329-339.

[12] Konstantinov, A. A., Siletsky, S., Mitchell, D., Kaulen, A., and Gennis, R. B.

(1997) The roles of the two proton input channels in cytochrome c oxidase from

Rhodobacter sphaeroides probed by the effects of site-directed mutations on

time-resolved electrogenic intraprotein proton transfer, Proc. Natl. Acad. Sci. U.S.A.

94, 9085–9090.

[13] Brzezinski, P., and Adelroth, P. (1998) Pathways of proton transfer in cytochrome

c oxidase, J. Bioenerg. Biomembr. 30, 99–107.

[14] Chang, H. Y., Hemp, J., Chen, Y., Fee, J. A., and Gennis, R. B. (2009) The



Acad. Sci. U.S.A. 106, 16169-16173.

[15] Soulimane, T., Buse, G., Bourenkov, G. B., Bartunik, H. D., Huber, R., and Than,

M. E. (2000) Structure and mechanism of the aberrant ba3-cytochrome c oxidase from

Thermus thermophilus, EMBO J. 19, 1766-1776.

[16] Preisig, O., Zufferey, R., Thöny-Meyer, L., Appleby, C. A., and Hennecke, H.






106

(1996) A high affinity cbb3-type cytochrome oxidase terminates the symbiosis specific

respiratory chain of Bradyrhizobium japonicum, J. Bacteriol. 178, 1532– 1538.

[17] Hemp, J., Han, H., Roh, J. H., Kaplan, S., Martinez, T. J., and Gennis, R. B.

(2007) Comparative genomics and site-directed mutagenesis support the existence of

only one input channel for protons in the C-family (cbb3 oxidase) of heme-copper

oxygen reductases, Biochemistry 46, 9963-9972.

[18] Arslan, E., Kannt, A., Thöny-Meyer, L., and Hennecke, H. (2000) The

symbiotically essential cbb3-type oxidase of Bradyrhizobium japonicum is a proton


[19] Koch, H. G., Winterstein, C., Saribas, A. S., Alben, J. O., and Daldal, F. (2000)

Roles of the ccoGHIS gene products in the biogenesis of the cbb3-type cytochrome c

oxidase, J. Mol. Biol. 297, 49–65.

[20] Toledo-Cuevas, M., Barquera, B., Gennis, R. B., Wikström, M., and



421–434.

[21] Zufferey, R., Preisig, O., Hennecke, H., and Thöny-Meyer, L. (1996) Assembly

and function of the cytochrome cbb3 oxidase subunits in Bradyrhizobium japonicum,

J. Biol. Chem. 271, 9114–9119.

[22] Kirichenko, A., Vygodina, T. V., Mkrtchyan, H. M., and Konstantinov, A. A.

(1998) Specific cation-binding site in mammalian cytochrome c oxidase, FEBS Lett.

423, 329-333.

[23] Pfitzner, U., Kirichenko, A., Konstantinov, A. A., Mertens, M., Wittershagen, A.,

Kolbesen, B. O., Steffens, G. C. M., Harrenga, A., Michel, H., and Ludwig, B. (1999)

Mutations in the Ca2+

-binding site of the Paracoccus denitrificans cytochrome c

107

oxidase, FEBS Lett. 456, 365-369.

[24] Riistama, S., Laakkonen, L., Wikstrom, M., Verkhovsky, M. I., and Puustinen, A.

(1999) The calcium binding site in cytochrome aa3 from Paracoccus denitrificans,


[25] Lee, A., Kirichenko, A., Vygodina, T., Siletsky, S. A., Das, T. K., Rousseau, D. L.,

Gennis, R. A., and Konstantinov, A. A. (2002) Ca2+

-binding site in Rhodobacter

sphaeroides cytochrome c oxidase, Biochemistry 41, 8886-8898.

[26] Mkrtchyan, H., Vygodina, T., and Konstantinov, A. A. (1990) Na+-induced

reversal of the Ca2+

-dependent red shift of cytochrome a. Is there a hydronium output

well in cytochrome c oxidase?, Biochem. Int. 20, 183-190.

[27] Kirichenko, A. V., Pfitzner, U., Ludwig, B., Soares, C. M., Vygodina, T. V., and

Konstantinov, A. A. (2005) Cytochrome c oxidase as a calcium binding protein.

Studies on the role of a conserved aspartate in helices XI-XII cytoplasmic loop in

cation binding, Biochemistry 44, 12391-401.

[28] Oh, J. I. and Kaplan, S., (2002) Oxygen adaptation. The role of the CcoQ subunit

of the cbb3 cytochrome c oxidase of Rhodobacter sphaeroides 2.4.1., J Biol Chem.

277, 16220-16228.

[29] Hemp, J., Christian, C., Barquera, B., Gennis, R. B., and Martinez, T. J. (2005)

Helix Switching of a Key Active-Site Residue in the Cytochrome cbb3 Oxidases,


[30] Thomas, P. E., Ryan, D., and Leven, W. (1976) An improved straining procedure

for the detection of the peroxidase activity of cytochrome P-450 on sodium dodecyl

sulfate polyacrylamide gels, Anal. Biochem. 75, 168-176.

[31] Berry, E. A., and Trumpower, B. L. (1987) Simultaneous determination of hemes

a, b, and c from pyridine hemochrome spectra, Anal. Biochem. 161, 1–15.

108

[32] Carrell, C. J., Ma, J. K., Antholine, W. E., Hosler, J. P., Mathews, F. S., and

Davidson, V. L. (2007) Generation of novel copper sites by mutation of the axial

ligand of amicyanin. Atomic resolution structures and spectroscopic properties,

Biochemistry. 46, 1900-12.

[33] Wikstrom, M., and Saari, H. (1975) A spectral shift of heme a induced by

calcium ions, Biochim. Biophys. Acta 408, 170-179.

[34] Saari, H., Pentilla, T., and Wikstrom, M. (1980) Interaction of Ca2+

and H+ with

heme a in cytochrome oxidase, J. Bioenerg. Biomembr. 12, 325-338.

[35] Thorndycroft, F. H., Butland, G., Richardson, D. J., and Watmough, N. J. (2007)

A New Assay for Nitric Oxide Reductase Reveals Two Conserved Glutamate

Residues Form the Entrance to a Proton-Conducting Channel in the Bacterial Enzyme,

Biochem. J. 401, 111-119.

[36] Flock, U., Thorndycroft, F. H., Matorin, A. D., Richardson, D. J., Watmough, N.

J., and Adelroth P. (2008) Defining the proton entry point in the bacterial respiratory

nitric-oxide reductase, J. Biol. Chem. 283, 3839-3845.

[37] Schmidt, B., McCracken, J., and Ferguson-Miller, S. (2003) A discrete water exit

pathway in the membrane protein cytochrome c oxidase, Proc. Natl. Acad. Sci. U.S.A.

100, 15539-15542.

STUDIES OF CYTOCHROME C OXIDASES FROM RHODOBACTER ...

Documents

Transcript of STUDIES OF CYTOCHROME C OXIDASES FROM RHODOBACTER ...