Principles of Bioinorganic Chemistry - 2004

Lecture Date Lecture Topic Reading Problems1 9/9 (Th) Intro; Choice, Uptake, Assembly of Mn+ Ions Ch. 5 Ch. 12 9/14 (Tu) Metalloregulation of Gene Expression Ch. 6 Ch. 23 9/16 (Th) Metallochaperones; Mn+-Folding, X-linking Ch. 7 Ch. 34 9/21 (Tu) Med. Inorg. Chem./MetalloneurochemistryCh. 8 Ch. 45 9/23 (Th) Mössbauer, EPR, IR Spectral FundamentalsCh. 9 Ch. 56 9/28 (Tu) Electron Transfer; Fundamentals Ch. 9 Ch. 67 9/30 (Th) Long-Distance Electron Transfer Ch. 10 Ch. 78 10/5 (Tu) Hydrolytic Enzymes, Zinc, Ni, Co Ch. 109 10/7 (Th) CO and Bioorganometallic Chemistry TBA Ch. 810 10/12 (Tu) Dioxygen Carriers: Hb, Mb, Hc, Hr Ch. 11 Ch. 911 10/14 (Th) O2 Activation, Hydroxylation: MMO, ToMOCh. 11 Ch. 1012 10/19 (Tu) Model Chemistry for O2 Carriers/ActivatorsCh. 12 Ch. 1113 10/21 (Th) Complex Systems: cyt. oxidase; nitrogenase Ch. 12 Ch. 1214 TBA Term Examination

Long-Distance Electron Transfer in Proteins

O1 O2 O3

etc

R1 R2 R3

O = oxidized formR = reduced form

Three ways to measure:

1. Self-exchange

2. Artificial donor-acceptor pairs3. Study of natural protein redox pairs

RedAz+ OxAz OxAz + RedAz

CuI CuII CuII CuI

k = 1.3 x 106 M-1 s-1 for azurin

Artificial Donor-Acceptor Pairs

Cytochrome c; Fe---Ru, ~12 Å

Method for Studying ET of Ru-Modified Proteins

[Ru(bpy)3]2+

flash photolysis

[Ru(bpy)3]2+* + RuIII–PFeIII

kQ

[Ru(bpy)3]3+

RuII–PFeIII + [Ru(bpy)3]3+

[Ru(bpy)3]2+

EDTA

kb back reaction

RuIII–PFeIII + [Ru(bpy)3]2+

kET kr

RuIII–PFeII

Notes

Monitor spectroscopically;

[Ru(bpy)3]2+* can react directly

with PFeIII in a reaction that is

fast compared to kET on protein.

Subtract from control experiment

with no modified surface His.Rate ~ 30 s-1, T-independent

Distance and Driving Force Dependencies of ET Rates

kET = (4π2 / )h T2DA ( ), FC where TDA is the tunneling matrix element

, and measures the electronic coupling of donor and acceptor FC is the

- , .Franck Condon factor and the other symbols have their usual meaning

T2D A = T

2DAexp(-β(R - Ro); at R = Ro, van der Waals contact

β is a medium effect parameter: related to electron "pathway"o

:Marcus Theory = (4FC πλ )kT -1/2 [-(-exp ΔGo - λ)2/4λkT

standard free energy of the reaction

Predicts k ET maximized

whenΔG o = - λ!!

reorganization energy

QuickTime™ and aTIFF (LZW) decompressor

are needed to see this picture.

Distance Dependence from the TDATerm for Reaction Center

Distance dependence from the TDA term for Ru-modified cytochrome c

β from the slope is 1.4 Å-1. Get a 10-fold decrease in rate for every 1.7 Å increase in distance

For comparison, β for ET in vacuum is 2.8 Å-1 and β for ET through covalent bonds is 0.7 Å-1

(thanks to Brian Crane for the plot)

Driving Force Dependence

Data are from ruthenium-modified cytochrome c derivatives (upper) and a series of covalently linked donor/acceptor compounds

The Mineral Springs in Bath, England,Source of Methylococcus capsulatus (Bath)

The Restutive Contents of the WATER’s Concoctive Power: Solution of gaffes, chaos of Salts and mineral effluvia of subterranean expiration. It cleanses the body from all blotches, scurvicial itchings and BREAKING OUTS WHATSOEVER!

e-

e-

CH4 + O2

+ 2e- + 2H+

(via MMOR)CH3OH +H2O

MMOH (Hydroxylation)

NADH +H+

NAD+

MMOR (Electron-Transfer)

MMOB Regulation of • Catalytic Efficiency • O2 Activation • Electron-Transfer

Structure and Function of the Protein Components of sMMO(Bath)

NMR Structure of theFd Domain of MMORMueller, Biochemistry, 41, 42-51 (2002)

samplecell

NADH or O2 solution

drive syringesmixing

chamber

Protein solution

low temperature dewarelectronictrigger

stop syringe

Protocol for Stopped-Flow/Freeze Quench KineticStudies of Reactions of the sMMO Proteins

-140 °Cisopentane bath

samplecell

0

5

10

15

20

400 450 500 550 600 650 700

RCT1CT2SQMC2

ε

(mM

-1

cm-1)

( )Wavelength nm

ox

0.0

0.050

0.10

0.15

0.200.25

0.30

0.35

0

0.01

0.02

0.03

0.04

0.05

0.06

0.01 0.1 1

A458

A625

, A740

( )Time s

458 nm

625 nm

740 nm

FADox

Fe2S2ox

NADH

FADox

Fe2S2ox

NADH

FADox

Fe2S2ox

NAD+

FADH-

Fe2S2ox

FAD sq

Fe2S2red

NADH

FAD sq

Fe2S2red

Rox 1MC 1CT 2CT SQ 2MC

NADH

NAD + NADHKd = 3.8 μM

350 s-1 190 s-1 100 s-1 30 s-1

Stopped-Flow Analysis of NADH Reduction of MMOR

Kopp, Blazyk, and Lippard, 1999

Single Wavelength Data with Simulations Spectra of Intermediates

Each trace is fit as a sum of exponentials giving rise to the reported rate constants.

0.00

0.02

0.04

0.06

0.00

0.10

0.20

0.30

0.0010.010.11 10100Time (s)

A625 & A

725

A458

625 nm (R)

625 nm (H + R)

458 nm (R)

458 nm (H + R)

725 nm (R)

0

5

10

15

20

400 500600 700

EoxInt-1Int-2Int-3Int-4Extinction (mM

-1cm-1)

Wavelength (nm)

FADox

Fe2S2oxox

ox

NADH

FADox

Fe2S2oxox

ox

NAD+

FADH-

Fe2S2oxox

ox

NADH

FAD(H-/sq)

Fe2S2(re/ox)(mv/ox)

ox

NADH

FADsq

Fe2S2redred

ox

Reaction of the MMOR:MMOH Complex with NADHIntra- and Intermolecular Electron-Transfer Steps

Gassner and Lippard, Biochemisry (1999)

NADH

FADsq

Fe2S 2ox

mv

ox

NADH

400 s-1 180 s-1

MMOR

MMOH

23 s-1

NAD +

NADH

106 s-1

Eox Int-1 Int-2 Int-3 Ered

NAD +

NADH

Int-4

12 s-1

CT1 CT2FADox MC1 FADhq

FAD

NADH

FAD

NADH

FAD FADH

NAD+

- FADH -

NAD+NADH

MMOR-FAD Reaction with NADH

Single wavelength (A) and diode array (B) optical stopped-flow experimentswere performed to examine the MMOR-FAD reaction with NADH.

0

2000

4000

6000

8000

10000

12000

400 450 500 550 600 650 700 750

Intermediates for FAD-ox + NADH Reaction

Extinction Coefficient/M

-1

cm-1

Wavelength/nm

CT1

CT2

FAD-hq

B

0.001 0.01 0.1 1

MC1CT1CT2FAD-hq

Concentration

Time/s

-0.004

0.000

0.004

0.008

0.001 0.01 0.1 1

Fit to Averaged Single WavelengthStopped-Flow Data for FAD-ox + NADH

Absorbance at 700 nm

Time/s

k1 = 166.6 ± 1.2 s-1

k2 = 94.4 ± 0.5 s-1

formation anddecay of CT2

A

These rate constants support the analysis with the full MMOR (Blazyk & Lippard, 2000)

Electron transfer between the two disconnected flavin and iron/sulfur domains occurs but at a

significantly reduced rate compared to the intact MMOR protein.

(Blazyk & Lippard, 2000)

F A D

h q

F A D H-

F e ( I I I ) / F e ( I I I )F do x

F dr e d

F A D H•

F e ( I I ) / F e ( I I I )

F A D

s q

B 2 B 3B 1

B 2B 1

B 3B 1

T h e r e a r e t w o o b s e rv e d

s t e p s f o r th e F A D - t o -F d

e l e c t r o n t r a n s f e r r e a c t i o n a t

2 5 . 0 ° C , w h i ch c a n b e f i t a s

p a r a l l e l o r s e q u e n t i a l

r e a c t i o n s .

I n t e r - D o m a i n E l e c t r o n T r a n s f e r

0

2 0 0 0

4 0 0 0

6 0 0 0

8 0 0 0

1 0 0 0 0

1 2 0 0 0

1 4 0 0 0

3 5 0 4 0 0 4 5 0 5 0 0 5 5 0 6 0 0 6 5 0 7 0 0 7 5 0

B 1

B 2

B 3

W a v e l e n g t h / n m

I n t e r m e d i a t e s f o r F A D + F d

R e a c t i o n

k1 = 0.080 s-1

k2 = 0.016 s-1

Electron Transfer from Flavin Domain to Fd Domain

Fe(III)/Fe(III)

Fe(II)/Fe(III)

Fe(II)/Fe(II)

+

Fd ox

Hmv

Hred

Fd red Fe(II)/Fe(III)

Fe(III)/Fe(III)

A2 A3A1

The reduced MMOR-Fdfragment is competent forelectron transfer into theoxidized MMOH diironactive site. The reactionoccurs in two kinetic steps.

Hox

?[ ]

Electron Transfer from MMOR-Fd to MMOH

N C[2Fe-2S]

19 frame-shifted residues

MW 15.0 kDa

MMOR Ferredoxin Fragment

The MMOR-Fd fragment wascreated from a failed mutagenesisreaction. It is significantly largerthan the designed Fd domain andcontains a C-terminal tail of 19frame-shifted residues.

0.14

0.16

0.18

0.20

0.22

0.001 0.01 0.1 1 10

Example of Fit to Single Wavelength Stopped-FlowData for Fd-red + MMOH-ox at 4 °C

Absorbance at 470 nm

Time/s

k1 = 36.97 ± 0.43 s-1

k2 = 8.01 ± 0.12 s-1

-2

0

2

4

6

8

10

0.0031 0.0032 0.0033 0.0034 0.0035 0.0036 0.0037

Arrhenius Plot for Electron Transfer fromMMOR-Fd Fragment to MMOH-ox

rate constant 1rate constant 2

ln (k/s

-1)

1/T (K-1)

Ea1

= 7.9 kcal mol-1

Ea2

= 7.1 kcal mol-1

For comparison, ET from 3-electron reduced MMOR to MMOH is characterized by apparent rate constants of 100 and 16 s-1.

•Three major metallic units transfer electrons in bioinorganic chemistry: iron-sulfur clusters; blue copper including the dinuclear CuA; and cytochromes (iron porphyrins).

•Electrons can transfer over long distances in ~10-15 Å hops . The rate depends on driving force, distance, and orientation of the reacting partners. Pathways are important ( > π > H-bonds according to theoretical models).

•Electron transfer within and between proteins is optimized to take advantage of the molecular switching stations. Included are organic units such as flavins and inorganic units such as iron-sulfur clusters, both used in the MMOR protein.

Summary - Points to Remember