Electron Transport and Oxidative Phosphorylation

An Overview

Biological oxidations are catalyzed by intracellular enzymes. The purpose of oxidation is to obtain energy. Electron Transport: Electrons carried by reduced coenzymes (NADH or FADH2) are passed sequentially through a chain of proteins and coenzymes (so called electron transport chain)to O2 . Oxidative Phosphorylation: Coupling e- Transport (Oxidation) and ATP synthesis (Phosphorylation) . It all happens in mitochondrion or at the inner mitochondrial membrane (eukaryotic cells)

mitochondrion
the mitochondrion contained the enzymes responsible for electron transport and oxidative phosphorylation

In inner membrane knobs

Impermeable to ions and most other compounds

Reduction Potentials
E0=standard reduction potential. The relative tendency to accept e-s and become Crucial equation: reduced.

Go' = -nF Eo'

Number of electrons = Eo'(acceptor) - Eo'(donor) transferred in the Faradays constant redox reaction (96485 J/volt/mole)

If Eo' is positive, an electron transfer reaction is spontaneous (Go' <0)

Fumarate+2H++2e- succinate FAD+ 2H++2e- FADH2 NAD++ 2H++2e- NADH+H+

0.031 0 -0.32

Succinate+FAD Fumarate+ FADH2 G' = - n F E' Eo' = Eo'(acceptor) - Eo'(donor) G =-2*96485*(0-0.031)= 5.98KJ/mol Succinate+ NAD+ Fumarate+ NADH+H+ G =-2*96485*(-0.32- 0.031)= 67.7KJ/mol

Removal of H across a C-C bond is not sufficiently exergonic to reduce NAD+,but it does yield enough energy to reduce FAD. Thats why succinate dehydrogenase uses FAD other than NAD+ as coenzyme.

Electron Carriers
The transfer of electrons is not directly to oxygen but through coenzymes There are 2 sites of entry NAD+ for electrons into the FMN electron transport chain: NAD+ or FAD
FeS FAD FeS ubiquinone Cyt b ubiquinone FeS Cyt c1 Cyt c Cyt a Cyt a3 1/2 O2

Both are coenzymes for dehydrogenase enzymes

Nicotinamide coenzymes: NAD+

X CONH2 XH2

H CONH2 + H+

N oxidised coenzyme + + NAD or NADP

N reduced coenzyme NADH or NADPH

Always a 2-electron reaction transferring 2 e- and 2 H+

The flavin coenzymes / flavoproteins

OH CH2 CH H3C N N N O
NH2 N OH CH2 CH H3C N N N O OH CH O OH CH CH2 O O P OH O O P OH O CH2 O N N N

OH CH O

OH CH CH2 O

O P OH OH

H3C

riboflavin monophosphate (flavin mononucleotide, FMN)

H3C

flavin adenine dinucleotide (FAD) OH

OH

FAD Always a 2-electron reaction transferring 2 e- and 2 H+

Oxidation and reduction of flavin coenzymes

O H C H3C H3C C C C H C C N CH2 HC HC HC H2C OH OH OH O O P OON C C N C NH C

O H C H3C
O H3C
-

O H C NH C H3C O H3C
-

H N C C C C N CH2 HC HC HC H2C OH OH

C C C

H N C C C C N CH2 HC HC HC OH OH

C NH C N H O

C C C H

C H

e +H

e +H

OH O O P OO-

OH O O P OO-

H2C

FMN

FMNH

FMNH2

it can accept/donate 1 or 2 e-. FMN has an important role in mediating e- transfer between carriers that transfer 2 e- (e.g., NADH) and those that transfer 1 e- (e.g., Fe+++).

Role of FMN mediating between 2e- & 1ecarriers: For example, when NADH donates electrons to the respiratory chain, the initial electron transfers are: NADH + H+ + FMN NAD+ + FMNH2 FMNH2 + Fe+++ FMNH + Fe++ + H+

Iron-sulfur Centers (clusters)

Iron-sulfur centers (Fe-S) are prosthetic groups containing 14 iron atoms Iron-sulfur centers transfer only one electron, even if they contain two or more iron atoms. E.g., a 4-Fe center might cycle between redox states: Fe+++3, Fe++1 (oxidized) + 1 e- Fe+++2, Fe++2 (reduced)

Cys S S Cys Cys S S Fe

S S Fe Fe S Fe

Fe S

S Cys Cys S S Cys

Fe S S Cys

Cys

Iron-Sulfur Centers

NAD+

Ubiquinone
O CH3O

FMN

Other names and abbreviations:

Coenzyme Q CoQ Q
FAD FeS

FeS ubiquinone Cyt b

CH 3 CH 3

CH3O O

ubiquinone FeS

(CH 2 CH

CH 2)nH

coenzyme Q

2 e- + 2 H+
OH CH3O CH 3 CH 3 CH3O OH (CH 2 CH C CH 2)nH

Most often n = 10 Free CoQ can undergo a 2 eoxidation/reduction: Q + 2 e- + 2 H+ QH2.

coenzyme QH2

When bound to special sites in respiratory complexes, CoQ can accept 1 e- to form a semiquinone radical (Q-).
O CH3O CH3 CH3 CH3O O (CH2 CH C CH3O OCH3 CH3 CH3O O (CH2 CH C CH2)nH

e-

CH2)nH

coenzyme Q

coenzyme Q -

e- + 2 H+
OH CH3O CH3 CH3 CH3O OH (CH2 CH C CH2)nH

coenzyme QH2

Coenzyme Q (CoQ, Q or ubiquinone) is lipid-soluble. It dissolves in the hydrocarbon core of a membrane.

the only electron carrier not bound to a protein.

it can accept/donate 1 or 2 e-. Q can mediate e- transfer between 2 e- that transfer and 1 ecarriers

Cytochromes
NAD+ FMN FeS FAD FeS ubiquinone Cyt b ubiquinone FeS Cyt c1 Cyt c Cyt a Cyt a3 1/2 O2

proteins that accept electrons from QH2 or FeS

Ultimately transfers the electrons to oxygen

Cytochromes
Cytochromes are electron carriers containing hemes . Hemes in the 3 classes of cytochrome (a, b, c) differ in substituents on the porphyrin ring.

Some cytochromes(b,c1,a,a3) are part of large integral membrane protein complexes. Cytochrome c is a small, water-soluble protein.

CH3 CH3 HC S CH2 protein

N H3C N
-

CH3 Fe N CH3 N CH S CH2 protein

OOC

CH2 CH2

CH2 CH2 COO-

CH3

Heme c

Heme is a prosthetic group of cytochromes. Heme contains an iron atom in a porphyrin ring system.

The heme iron can undergo 1 e- transition between ferric and ferrous states: Fe3+ + e Fe2+ Copper ions besides two heme A groups (a and a3) act as electron carriers in Cyta,a3 Cu2++e- Cu+

Electron carriers
NAD+, flavins and Q carry electrons and H+ Cytochromes and non-haem iron proteins carry only electrons

NAD+ FAD undergoes only a 2 e- reaction; cytochromes undergo only 1e- reactions FMN Q undergoes 1e- and 2 e- reaction

Electron Transport chain (respiratory chain)

The electron transport chain in the inner mitochondrial membrane can be isolated in four proteins complexes(I, II, III, IV). A lipid soluble coenzyme (Q) and a water soluble protein (cyt c) shuttle between protein complexes Electrons transfer through the chain - from complexes I and II to complex IV

The electron transport chain Mitochondrial Complexes

Mitochondrial Complexes
NAD+ FMN

I
FeS FAD FeS ubiquinone Cyt b

NADH Dehydrogenase

II
Succinate dehydrogenase
ubiquinone

Cytochrome Oxidase
FeS Cyt c1 Cyt c Cyt a Cyt a3 1/2 O2

III
CoQ-cyt c Reductase

IV

Support for this order of events

1. Energetically favorable. electrons pass from lower to higher standard reduction potentials . Spectra: the absorption spectrum for the 2. reduced carrier differs from that of its oxidized form. carriers closer to oxygen are more oxidized. 3. Specific inhibitors. Those before the blocked step should be reduced and those after be oxidized.

4. Assay of individual complexes.

NADH can reduce complex I but not the other complexes.

Order and Reduction Potentials

NAD+ FMN FeS FAD FeS ubiquinone Cyt b ubiquinone FeS Cyt c1 Cyt c

-0.32

-0.3

+0.045 +0.077

+0.03

+0. 29
Cyt a

+0. 55
Cyt a3 1/2 O2

+0. 22

+0. 25

+0.82

Drugs that inhibit the ETC

NAD+

Amytal I rotenone
FAD FeS

FMN FeS ubiquinone Cyt b

Rotenone helps natives of the Amazon rain forest catch fish!

II
Antimycin A
ubiquinone

CN- CO
binding tightly to the ferric form (Fe3+) of a3

FeS

Cyt c1

Cyt c

Cyt a

Cyt a3 1/2 O2

III

IV

When the chain is blocked, electron carriers will be in a reduced state before the block point and in an oxidized state after it. This can easily be monitored using difference spectra.

Inhibitors and Artificial Electron Acceptors

Rotenone amytal

Antimycin A

CN-,CO

Methylene blue +0.01 Ferricyanide+0.36

+ H

Transport

Complex I, III, IV drive H+ transport from matrix to the cytosol When e- flow through, which creates proton gradient(electrochemical potential) across the inner membrane Complex I and Complex IV : The mechanism of H+ transport is still not known. The mechanism of H+ transport in Complex III is Q cycle.

Matrix
H+ + NADH NAD+ + 2H+ 2H+ + O2 H2O

2 eQ

III IV

++
4H+ 4H+

cyt c

2H+

Intermembrane Space

4H+ are pumped per 2e- passing through complex III. The H+/e- ratio is less certain for the other complexes: probably 4H+/2e- for complex I; 2H+/2e- for complex IV.

Q Cycle :The mechanism of H+ transport in Complex III

matrix Q 2 H+ Q.cyt bH
Complex III

QH2

QH2

cyt bL Q e- Q.

QH2 eFe-S 2 H+ cyt c1 cyt c

intermembrane space

1.Electrons are transported along the inner mitochondrial membrane, through a series of electron carriers 2.Protons (indicated by + charge) are translocated across the membrane, from the matrix to the intermembrane space 3.Oxygen is the terminal electron acceptor, combining with electrons and H+ ions to produce water 4. As NADH delivers more H+ and electrons into the ETS, the proton gradient increases, with H+ building up outside the inner mitochondrial membrane, and OH- inside the membrane.