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 11/4 (Th) Term Examination

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

Example of fit to single wavelength stopped-flow data for the reactionFd(1-120)red + MMOHox at 4 °C

For comparison, ET from 3-electron reduced MMOR to MMOH at 4 °C is characterized by apparent rate constants of 82 s-1 and 17 s-1.0.08

0.10

0.12

0.14

0.16

0.18

0.20

0.001 0.01 0.1 1 10

Time (s)

k1 = 34.9 ± 0.5 s-1

k2 = 10.3 ± 0.3 s-1

0

20

40

60

80

100

120

140

280 290 300 310

rate constant 1rate constant 2

Temperature (K)

0

1

2

3

4

5

6

7

0.0032 0.0033 0.0034 0.0035 0.0036 0.0037

rate constant 1rate constant 2

1/T (K-1)

Arrhenius plot ET theory analysis

ln k = ln A – (Ea/R)(1/T)Ea1 = 8.6 kcal mol-1

Ea2 = 6.6 kcal mol-1

€

kET = k0e−β (r−r0 )e[−(ΔG°+λ )2 / 4λRT ]

HAB(1) = 0.8 cm-1

λ1 = 1.5 eVr1 = 11.3 Å

HAB(2) = 0.1 cm-1

λ2 = 1.3 eVr2 = 14.3 Å

•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

Hydrolytic Enzymes, Zinc and other Metal Ions

PRINCIPLES:

•M(OH)n+ centers supply OH- at pH 7 by lowering water pKa

•Mn+ serves as general Lewis acid, activating substrates•Rate acceleration occurs by internal attack within coord. sphere•Protein side chains greatly assist assembly of transition state•Carboxylate shifts can occur, especially at dimetallic centers•Electrostatic interactions predominate•Non-redox active metal ions often but not universally used

Illustrating the Principles:

•Carboxypeptidase, carbonic anhydrase - delivering hydroxide•Alcohol dehydrogenase: an oxidoreductase•Dimetallic metallohydrolases: are two metals better than one?

Carboxypeptidase A: A Hydrolytic Zinc Enzyme

Reaction catalyzed:

R–CH–C(O)–NH–R’

NH2R’’

R–CH–CO2- + +NH3–R’

NH2R’’Cleaves C-terminal peptide bonds; prefers aromatic residues.

Active site contains a single catalytic zinc, essential for activity. The glutamate can undergo a carboxylate shift. Thermolysin has a similar active site; it is an endopeptidase.

Carboxypeptidase A structure with the inhibitor glycyl-L-tyrosine bound at the active site. Note hydrogen bonds to key residues in the active site that position the substrate moiety for bond scission.

Catalytic Mechanism for Carboxypeptidase ASummary of events:1. Substrate binds; orients by the terminal carboxylate.2. Deprotonate bound H2O.3. Polarize scissile bond by Arg127.4. Bound OH- attacks peptide C(O).5. Form tetrahedral transition state.6.Lose 2 peptide fragments and recycle the enzyme.

Principles illustrated:1. Zinc serves as template.2.Metal supplies cleaving reagent, OH-, and organizes key groups.3. Chemistry achieved at neutral pH! Kcat ~ 100 s-1 .

Carbonic Anhydrase, the First Known Zn Enzyme

Reaction catalyzed:

CO2 + H2O H2CO3 ~ 106 s-1

Note: Rate 10-2 s-1 at pH 7; kf 106 s-1 in active site.Paradox: The reverse reaction is diffusion controlled, with kr ~ 1011 M-1 s-1

Thus kf ≤ 104 s-1. So how can the turnover be 106 s-1 ? Answer: Facilitated diffusion of protons by buffer components bound to the enzyme.

PZn(OH2)2+ PZn(OH)+ + H+Keq = 10-7 M = kf/kr

Carbonic Anhydrase

Possible Carbonic Anhydrase Mechanism

Alcohol Dehydrogenase, an Oxidoreductase

Reaction catalyzed:RCH2 OH + NAD+ RCHO + NADH + H+

Enzyme contains two 40 kDa polypeptides, each with 2 Zn2+centers in separate domains. One zinc is structural, the other catalytic.

Catalytic zinc is 20 Å from the surface, near the nicotinamide binding region. This center is not required for NAD + cofactor binding. Alcohol substate DO require zinc and bind directly to the metal center, displacing the coordinated water.

Schematic Diagram

NAD+ binding to the active site of LADH, with specific, well-positioned amino acid side chains holding it in place. Ethanol is shown bound to the zinc, displacing water. The system is set to undergo catalysis.

Hydride Transfer Mechanism

N

H2N O

R' +

OZn

H

RH

H2O

N

H2N O

R' ..

H

H

+ RCHO +

H2OZn

Note hydride transfers from -C of alcohol to nicotinamide ring.

Dinuclear Metalloenzymes

Peptide hydrolases: Methionine aminopeptidase (Zn2 or Co2)Leucine aminopeptidase (Zn2)

Phosphoester hydrolases:Ser/Thr phosphatases (Fe/Zn or Fe/Fe)Alkaline phosphatase (Zn2)Nuclease P1 (Zn2)Inositol Monophosphatase (Mg2)RNase (Mn2 and Mg2)DNA polymerase I (Mg2)

Other metallohydrolases:Arginase (Mn2, Co2)Urease (Ni2)β- (Lactamase Zn2)

(Xylose isomerase Mg2):Isomerase

- Redox active dinuclear:Metalloenzymes

(Methane monooxygenase Fe2) (Tyrosinase Cu2)

(Catalase Mn2)

pKa Values of Metal-Bound Water for CommonMetal Ions in Aqueous Solution

pH1 2 3 4 5 6 7 8 9 10 11 12 13 14

Fe3+-OH

Cu2+-OH

Zn2+-OH

Co2+-OH

Ni2+-OH

Mn2+-OH

Mg2+-OH

M OH2 M OH H+pKa

pKa

Barnum, D. W. Inorg. Chem. 1983, 22, 2297.

+

Dimetallics can move the value into the physiological range near pH 7

MB

OC

O

MA

R

N S

MB

OC

O

MA

R

S

MB

OC

O

MA

R

N S

MB

OC

O

MA

R

N SMB

OC

O

MA

R

N S

Modes of Substrate (S) Attack by an Activated Nucleophile (N) at a Carboxylate-Bridged Dimetallic Center

A EB C D

N:: :: :

Advantages of Carboxylate-Bridged Dimetallic Centersin Chemistry and Biology

+H3N NH

NH2

CO2-

NH2+

+H3N NH3+

CO2- H2N NH2

O+

L-Ornithine

H2O +

L-Arginine Urea

Structure and Chemistry of Arginase

Mn2+ O

Mn2+

O

O

O

Asp-232

O O

Asp-124

O

O

Asp

OAsp-128

NNH

His

NHN

His

H

Active Site of Arginase

NH

NH2

NH2

O

O

Mn2+ OMn2+

NH

NH2

NH2

O

O

OMn2+ Mn2+

H

NH2

O NH2

NH2

O

OMn2+

Mn2+

Mn2+

H2O

Mn2+

III

L-Ornithine

II

IV

H2O

Urea

H+

L-Arginine

Postulated Catalytic Mechanism for Arginase

I O OO

OH

OO

Asp 124

Asp 128

Christianson, 1996

Principles illustrated: the dimetallic affords hydroxide; the substrate is positioned by residues in the active site; the dimetallic stabilizes the urea leaving group; redox inactive metal; electrostatics

Alkaline Phosphatase; a Dizinc(II) Center Activates the Substrate

1. The substrate binds to the dizinc center; a nearby Arg also helps activate it.2. A serine hydroxyl group attack the phosphoryl group, cleaving the ester. The phosphate is transferred to the enzyme, forming a phosphoryl-serine residue.3. Hydrolysis of this phosphate ester by a zinc-bound hydroxide com-pletes the catalytic cycle.

This mechanism is supported by studies with chiral phosphate esters (ROP18O17O16O)2-; there is no net change in chirality at phoshorus.

1.

2.

3.

The Dinickel(II) Metalloenzyme Urease

History of Urease

1926, Sumner crystallizes urease

1975, Blakeley and Zerner discover that urease is a dinickel enzyme

1995, Hausinger and Karplus determine X-ray structure; unusual active site

Ni Ni

N

N

N

N

N

NN

N

N

OO

O

O

O

Urea Hydrolysis

H2N

O

NH2 H2N

O

OH

H2Ourease + NH3

NH3 + H2O

N

NH

N

HN

NHN

NHN

Ni1O O

Ni2

HN

Lys220*

OH(2)OH2 O

H2OO Asp363

His137

His139

Figure 1. Barrios & Lippard

NNH

N

HN

NHN

NHNNi1

O O

Ni2

HNLys220*

OO ONH2

O Asp363

His137

His139N

NH

N

HN

NHN

NHNNi1

O O

Ni2

HNLys220*

SHO O

O Asp363

His137

His139

P

NH2His249

His275His275

His249

His275

His249

Native and Inhibited Urease from B. Pasteurii

Benini et. al. Structure 1999, 7, 205-216.Benini et. al. JBIC 1998, 3, 268-273.

Native urease, 2.0 Å resolution

β- , Mercaptoethanol inhibited urease1.65 Å resolution

, 2.0 DAP inhibited urease Å resolution

NiNi

OH

NiNi

OH O

NH2H2N

NiNi

O O

NH2

O

NH2H2N

Proposed Mechanism of Urea Hydrolysis

O

NH2H

O

NH2

O

NHCH3H2N

O

NHOHH2N

O

NHOHHOHNOther urease substrates:

H2O

CO2 + (2)NH3

NiNi

OH

NiNi

OH O

NH2H2N

NiNi

OH2

O

NH2H2N

O

C

N

Alternative Mechanism of Urea Hydrolysis

H2O

CO2 + (2)NH3

Enzymatic Catalysis of Urea Decomposition: Elimination or Hydrolysis?

Guillermina Estiu and Kenneth M. Merz, Jr.pp 11832 - 11842

•Both mono- and dimetallic centers lower the pKa value of bound water, allowing hydroxide to be delivered at pH 7.

•Coordination of the leaving group portion of the substrate to a metal ion activates the substrate for nucleophilic attack.

•Residues not coordinated but in the second coordination sphere can participate directly (serine in phophatases) or indirectly (arginine in alcohol dehydrogenase) in substrate attack, orientation, and/or activation.

•Carboxylate shifts facilitate substrate binding, activation.

•Redox inactive metal ions (Zn2+, Ni2+, Mn 2+, Co2+) preferred.

Summary - Points to Remember