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ATP

•  Universal carrier of free energy

•  Provides energy for •  Mechanical work •  Chemical work •  Ionic work

Oxidative Phosphorylation (Aerobic Respiration)

Oxidative phosphorylation •  is aerobic (i.e., in O2) •  is a stepwise process

Electron transfer chain of oxidative phosphorylation

General principles of redox reactions An oxidation-reduction (redox) reaction involves

an electron donor and an electron acceptor.

The redox potential expresses the tendency of an electron donor to reduce its conjugate acceptor.

Under standard conditions (25oC, pH 7, [donor]=[acceptor]=1 M), the redox potential is Eo’

Eo’ is measured relative to the standard hydrogen electrode.

Fe2+ + Cu2+ Fe3+ + Cu+

e- donor e- acceptor oxidized donor

reduced acceptor

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Reduction potentials Compounds with a large negative Eo are strong reducing agents.

Compounds with a large positive Eo are strong oxidizing agents.

Redox pair Eo’ (V)

NAD+ / NADH

FMN / FMNH2

Cytochrome b Fe3+/Fe2+

1/2 O2 / H2O

-0.32

-0.22

+0.07

+0.82

Coupled oxidation-reduction reactions

Res

pira

tory

ele

ctro

n ca

rrie

rs

NAD+

FMN

Fe-S centers

Coenzyme Q

Cyto b (Fe3+)

Fe-S centers

Cyto c (Fe3+)

Cyto a (Fe3+)

Cyto a3 (Fe3+)

O2

Cyto c (Fe3+)

- 0.32

- 0.30

+ 0.04

+ 0.07

+ 0.23

+ 0.29

+ 0.55

+ 0.82

+ 0.25

Eo’

NAD+ / NADH

FMN / FMNH2

Fe3+S / Fe2+S

Fe3+S / Fe2+S

H-Fe3+ / H-Fe2+

H-Fe3+ / H-Fe2+

H-Fe3+ / H-Fe2+

H-Fe3+ / H-Fe2+

CoQ / CoQH2

O2 / H2O

H-Fe3+ / H-Fe2+

Redox couples

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Mitochondrial electron transport chain

The electron transport chain conducts a series of oxidation/reduction reactions.

The components of the respiratory chain are flavoproteins, ubiquinone molecules, and cytochromes

Final e- acceptor is molecular oxygen

2 e¯

Citric acid cycle

Mitochondrial electron transport chain

bH bL

2Fe-2S

c1

Cyto b

Cyto c1

Cyto c

Q 2Fe-2S

QH2

FMN

4Fe-4S NADH

QH2

matrix

intermembrane space CuA

a

a3

CuB

1/2 O2 +

2 H+

H2O

Complex I: NADH-Q reductase NADH + H+

NAD+

FMN

FMNH2

Fe2+S

Fe3+S

CoQ

CoQH2

Q 2Fe-2S

QH2

FMN

4Fe-4S

NADH

QH2

matrix

intermembrane space

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Redox states of coenzyme Q

Reduced form of coenzyme Q

(QH2, ubiquinol)

H3CO

H3CO

OH

OH

CH3

CH2

CH

CH2C

CH3H

10

H3CO

H3CO

O

OH

CH3

R

H3CO

H3CO

O

O

CH3

R

e- + H+ e- + H+

Semiquinone intermediate

(QH•)

Oxidized form of coenzyme Q

(Q, ubiquinone)

Complex III: cytochrome bc1

QH2

QH• Cyt 2+ b Fe2+S

Fe3+S Cyt 3+ b

Q

QH•

Cyt 3+ c1

Cyt 2+ c1

Cyt 2+ c

Cyt 3+ c

N

N N

N

OH OHO O

Fe

matrix

intermembrane space

bH

bL

2Fe-2S c1

Cyto b

Cyto c1

Cyto c

QH2

Complex IV: cytochrome oxidase

Cyto c

matrix

intermembrane space CuA

a

a3

CuB

1/2 O2 +

2 H+

H2O

Cyt 2+ a3

Cyt 3+ a3

Cyt 3+ a

Cyt 2+ a

Cyt 2+ c

Cyt 3+ c H2O

1/2 O2

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Inhibitors of mitochondrial electron transport

Blocking electron transfer by any one of these inhibitors steps electron

flow from substrate to oxygen.

These inhibitors decouple the oxidation-reduction pathway in

electron transport.

Amytal Rotenone

Antimycin A

CN- CO

sodium azide

H+

H

H+ H+ H+

Mitochondrial electron transport chain

bH bL

2Fe-2S

c1

Cyto b

Cyto c1

Cyto c

Q 2Fe-2S

QH2

FMN

4Fe-4S NADH

QH2

matrix

intermembrane space CuA

a

a3

CuB

1/2 O2 +

2 H+

H2O

ATP

ADP + Pi

For 1 mol NADH oxidized, 3 mol ATP produced

Chemiosmotic Coupling Theory

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F1FO synthase 3 H+ ATP

ADP + Pi

ATP

ADP + Pi

When electrochemical H+ gradient is favorable, F1FO complex catalyzes ATP synthesis.

If no membrane potential or pH gradient exists to drive the forward reaction, Keq favors the reverse reaction (ATP hydrolysis).

3 H+

F1FO synthase

F1 subunit

•  present with stoichiometry α3, β3, γ, δ, and ε •  α and β subunits (513 and 460 residues in

E. coli) are homologous to one another •  3 nucleotide-binding catalytic sites at α/β

interface, but involving β residues

F0 subunit

•  present with stoichiometry a, b2, and c10

F1

F0

α β

α α β

β γ

Binding change mechanism

•  Proposed by Paul Boyer (Nobel Prize in Chemistry 1997)

•  Existence of 3 catalytic sites in F1 domain

•  An irregularly shaped ‘shaft’ linked F0 rotation relative to the 3 β subunits.

•  Rotation is drive by flow of H+ through F0

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Binding change mechanism

•  Conformation of each β subunit changes sequentially, as it interacts with rotating shaft.

•  Each β subunit is in a different catalytic stage at any time.

ATP

ATP ATP open

ATP

ATP

ADP + Pi

ADP + Pi

ADP + Pi tight

loose

F1FO synthase

•  John Walker’s group solved the F1 structure by diffraction (Nobel Prize in Chemistry 1997)

•  Crystal structure of F1 with the central stalk supports the binding change model

•  The γ subunit has a bent helical loop that constitutes a shaft within the ring of α and β subunits

•  3 β subunits were found to differ in conformation and bound ligand

Rotation of the γ shaft relative to the ring of α and β subunits directly observed, by attaching fluorescent-labeled actin filament to the γ subunit.

Noji et al. 1997 Nature 386, 299

The rotation was found to be ATP-dependent. Rate is 100 Hz (revolutions/s)

F1FO synthase

http://www.res.titech.ac.jp/~seibutu/main_.html