Oxidative phosphorylationBiochemistry, 4th edition, RH Garrett & CM Grisham, Brooks/Cole (Cengage); Boston, MA: 2010

pp 592-629

Instructor: Kirill Popov

1. The mitochondrion

2. Electron transport

3. Oxidative phosphorylation

4. Heat, oxidative stress, etc.

ATP synthase

Cristae

Ribosomes

Porin channels

Outer membrane

freely permeable tosmall molecules and ions

Inner membrane

Impermeable to mostsmall molecules and ionsIncluding H+

Contains:• Respiratory electron carriers• ADP/ATP translocase• ATP synthase• Other membrane transporters

Matrix

Contains:• Pyruvate dehydrogenase complex• Citric acid cycle enzymes• Fatty acid β-oxidation enzymes• Amino acid oxidation enzymes• DNA, ribosomes• Other enzymes and metabolites

Biochemical anatomy of a mitochondrion

)10(

CH3

CH2 C CH2

CH3

CH2CH3O

CH3O

O

O

H

H+ + e−

Ubiquinone (Q)(fully oxidized)

H

CH3

CH3O

CH3O

O

OR

H

•

CH3

CH3O

CH3O

O

O

RH

H+ + e−

Semiquinone radical(•QH)

Ubiquinol (QH2)(fully reduced)

Iron protoporphyrin IX(in b-type cytochromes)

Heme A(in a-type cytochromes)

Heme C(in c-type cytochromes)

N

N

NN

H3C CH

CH3

CH2CH2COO-

CH2CH2COO-H3C

H3C

CH

CH2

H2C

Fe

N

N

NN

H3C CHCH3

CH3

CH2CH2COO-

CH2CH2COO-H3C

H3C

CH3CH

S

S Cys

Cys

Fe

Fe

N

N

NN

H3C CH

CH3

CH2CH2COO-

CH2CH2COO-CHO

H3C

CH

CH2

CH2

OH

H3C

CH3CH3CH3

Prosthetic groups of cytochromes

50

Rel

ativ

e lig

ht a

bsor

ptio

n (%

)

Wavelength (nm)

400 500 600300

100

0

γ

Oxidizedcyt c

Reducedcyt c

α

β

Adsorption spectra of cytochrome c

Fe SCys

Fe

Fe

FeFeFe

Fe

SS

S

S

S S

S

S

S

S

S

S

S

S

S

S

SCys

CysCys

Cys Cys

Cys

Cys

Cys

Cys

Cys

Cys

Protein

Iron-sulfur centers

Standard Reduction Potentials of Respiratory Chain and Related Electron Carriers

Redox reaction (half-reaction) E'° (V)

2H+ + 2e− → H2 -0.414

NAD+ + H+ + 2e− → NADH -0.320

NADP+ + H+ + 2e− → NADPH -0.324

NADH dehydrogenase (FMN) + 2H+ + 2e− → NADH dehydrogenase (FMNH2) -0.30

Ubiquinone + 2H+ + 2e− → ubiquinol 0.045

Cytochrome b (Fe3+) + e− → cytochrome b (Fe2+) 0.077

Cytochrome c1 (Fe3+) + 2e− → cytochrome c1 (Fe2+) 0.22

Cytochrome c (Fe3+) + 2e− → cytochrome c (Fe2+) 0.254

Cytochrome a (Fe3+) + 2e− → cytochrome a (Fe2+) 0.29

Cytochrome a3 (Fe3+) + 2e− → cytochrome a3 (Fe2+) 0.35

1/2O2 + 2H+ + 2e− → H2O 0.8166

Separation of functional complexes of the respiratory chain

The Protein Components of the Mitochondrial Electron-Transfer Chain

Enzyme complex/protein Mass (kDa) Number of subunits* Prosthetic group(s)

I NADH dehydrogenase 850 43 (14) FMN, Fe-S

II Succinate dehydrogenase 140 4 FAD, Fe-S

III Ubiquinone:cytochrome c oxidoreductase 250 11 Hemes, Fe-S

Cytochrome c# 13 1 Heme

IV Cytochrome oxidase 160 13 (3-4) Hemes, CuA, CuB

*Numbers of subunits in the bacterial equivalents in parentheses.#Cytochrome c is not part of an enzyme complex; it moves between Complexes III and IV as a freely soluble protein.

Flavoprotein 4

Flavoprotein 1

Flavoprotein 2

Glycerolphosphatedehydrogenase,

FAD,Fe-S centers,

Flavoprotein 3

NADH dehydrogenase,FMN,

Fe-S centers

Succinate dehydrogenase,FAD,

Fe-S centers,b-type heme

Cytochrome bc1 complex,2 b-type hemes,

Rieske Fe-S centerc-type heme (cyt c1),

Electron-transferringf lavoprotein, FAD,

Fe-S centers,

Cytochrome cCytochrome aa3 complex,

2 a-type hemes,Cu ions

Complex IV

Complex II

Complex III

UQ/UQH2pool

Complex I

Fatty acyl-CoA dehydrogenase

NADH coenzyme Qoxidoreductase

Coenzyme Q-cytochrome coxidoreductase

Succinate-coenzyme Qoxidoreductase

Cytochrome c oxidase

H2O1/2 O2

Pathways in the mitochondrial electron transport

Complex IV

NA

D+ /

NA

DH

FMN

(Fe/

S)N

2(F

e/S

)N3

(Fe/

S)N

4(Fe/

S)N

1

Complex I

Complex IIComplex III

Rie

ske

Fe/S

(Fe/

S)S

1

(Fe/

S)S

3FA

D

Fum

/Suc

c

UQ

10

b Lb H

c 1 c

Cu A

a 3

a

-200

+200

+400

+600

0

-400

E(m

V)

Electrons move downhill

FMN

Fe-S

NAD+

NADH + H+

Intermembranespace (P side)

Matrix (N side)

Matrix arm

2e−

Seriesof Fe-Scenters

N-2Q

QH22e−

2H+

4H+

Membranearm

Complex I

NADH:ubiquinon oxidoreductase (Complex I)

Q

Intermembranespace (P side)

Matrix (N side)2H+

QH2

Fe-S

FAD

2e−

Seriesof Fe-Scenters

2e−

b-type heme

Substratebinding

site

Ubiquinone

Succinate Fumarate

α-Ketoglutarate

Malate

Oxaloacetate

CitrateIsocitrate

Succinyl-CoA

Acetyl-CoA

Krebs cycle

Succinate dehydrogenase (Complex II)

Cytochrome b

Heme

Cytochromec1

Cytochromec

2Fe-2Scenter

Rieske iron-sulfur protein

Intermembranespace (P side)

Matrix (N side)

c1

bL

bH

Qp

QN

Cytochrome bc1 complex (Complex III)

Intermembranespace (P side)

Matrix (N side)

bL

bH

bL

bH

Cyt c1

Cyt cCyt c

Cyt c1

QH2

2H+

2H+2H+

Q

Q Q•Q•

QH2QH2

Oxidation of first QH2 Oxidation of second QH2

Q

Fe-S Fe-S

QH2 + Cyt c1 (oxidized) → Q•− + 2HP

+ + Cyt c1 (reduced)QH2 + Q•− + Cyt c1 (oxidized) →

QH2 + 2HP+ + Q + Cyt c1 (reduced)

Net equation:QH2 + 2 Cyt c1 (oxidized) + 2HN

+ → Q + 2 Cyt c1 (reduced) + 4HP+

The Q cycle

4H+

(substrate)

Intermembranespace(P side)

2H2O2H+

(pumped)

4Cyt c4e-

O2

2H+

CuA

CuB

a

a3

I

IIIII

Fe-Cu center

Path of electrons through comlex IV

CuFe

Cu

Cu

Cu

Cu

Cu

Fe

Fe

Fe

Fe

Fe

O2

O

O

O−

O

2+

O

O−

2H+

H HH+ H+2H2O

1+

1+

2+

2+

2+

2+

2+3+3+

3+

4+

1st e−

2nd e−

3rd e−4th e−

next cycle

Reaction sequence for the reduction of O2 by the cytochrome c oxidase

The flow of electrons and protons through the respiratory chain (proton-motive force and chemiosmotic model)

IntermembraneSpace (P side)

Matrix (N side)II

I

IV

ADP +Pi ATP

Fo

F1

NADH + H+ NAD+ Succinate Fumarate

H2O1/2O2 + 2H+

4H+ 4H+

Q

Cyt c

III

2H+

Cyt c

H+

+ + + + + + + + + + + + + +

-------

Chemicalpotential

ΔpH(inside

alkaline)

ATPsynthesisdriven by

proton-motiveforce

Electricalpotential

Δψ(inside

negative)

ΔG = RT ln (C2/C1) + ZFΔψ= 2.3RT ΔpH + FΔψ

H+

H+

H+

H+

H+

H+

H+

H+

OH−

OH−

OH−

OH−

OH−

OH−

OH−

[H+]P = C2 [H+]N = C1

N sideP side

Protonpump

H2O

P ATP

ATPP

H+H+

Enzyme bound

In the absence of a proton gradient:

In the presence of a proton gradient:

is releasedADP +

ADP + ADP +-

18O P OH

O-

O18OH2

Catalytic mechanism of F1

Mitochondrial ATP synthase complex

Rotation of Fo and γ

αβ ATP

ADP + Pi

α

C10a

b

γ

His residuesHis residues

Ni complex

Actin filament

Avidin

ε

Rotation of Fo and γ

N side

P side

C10a

b2

α β

γ

ε

ATP

ADP + Pi

δ β

H+

H+

F1

Fo

A model of the FoF1 complex, a rotating molecular motor

ATP

ADP+ Pi

ATP

ATP

ATP

ATP

ATP

ADP+ Pi

ADP+ Pi

α

β

α

α

α

α

α

α

α

α

β

β

β

β

β

β

β

β

3 HP+

3 HN+

3 HN+

3 HN+

3 HP+

3 HP+

Binding-change model for ATP synthase

The P/O ratio is an index of the efficiency of coupling

P/O ratio: number of molecules of Pi incorporated (=ATP synthesized) per atom of oxygen consumed (or pair of electrons being carried through the chain).

Measurements: oxygen consumption during complete phosphorylation of a fixed amount of ADP after addition of either an NAD+-linked substrate or FAD-linked substrate.

P/O 2.5 for NADHP/O 1.5 for FADH2

Intermembranespace

Matrix

Adeninenucleotidetranslocase(antiporter)

H+ H+

H+

ADP3-

ATP4-

ATPsynthase

Phosphatetranslocase(symporter)

ADP3-

ATP4-

H+

H2PO4− H2PO4

−

Adenine nucleotide and phosphate translocases

Matrix

Q III

Glycerol 3-phosphate

Dihydroxyacetonephosphate

NADH + H+NAD+

Glycolysis

cytosolicglyceol 3-phosphate

dehydrogenase

mitochondrialglyceol 3-phosphate

dehydrogenase

FADFADH2

CH2OH

C

CH2 O

O

P

CH2OH

CHOH

CH2 O P

Glycerol 3-phosphate shuttle

Intermembranespace

Matrix

NADH + H+

α-Ketoglutarate

Malate Malate

OxaloacetateOxaloacetate

Aspartate Aspartate

Glutamate Glutamate

α-Ketoglutarate

Malateα-ketoglutaratetransporter

Glutamate-aspartatetransporter

malatedehydrogenase

aspartateaminotransferase

aspartateaminotransferase

malatedehydrogenase

NAD+ NAD+

H+ + NADH

Malate-aspartate shuttle

ATP Yield from Complete Oxidation of GlucoseProcess Direct product Final ATP

Glycolysis 2 NADH (cytosolic)2 ATP

3 or 5*2

Pyruvate oxidation (two per glucose) 2 NADH (mitochondrial matrix) 5

Acetyl-CoA oxidation in citric acid cycle (two per glucose)

6 NADH (mitochondrial matrix)2 FADH2

2 GTP

1532

Total yield per glucose 30 or 32

*The number depends on which shuttle system transfers reducing equivalents into the mitochondrion.

Intermembranespace

Matrix

Uncouplingprotein

(thermogenin)

Cyt c

H+

II

H+

H+

Heat

I

IV

III

ADP +Pi

ATP

Fo F1

Heat generation by uncoupled mitochondria

Innermitochondrialmembrane

I

IVIII

Cyt c

Nicotinamidenucleotidetranshydrogenase

O2

•O2−

•OHNADH NAD+

NADPH

NAD+ NADP+ H2O2

superoxidedismutase

glutathioneperoxidase

H2O

GSSG

2 GSH

2 GSH

glutathionereductase

protein thiolreduction

GSSG

inactive

oxidativestress

active

EnzS

SSH

SH

Q

ROS formation in mitochondria and mitochondrial defenses

HIF-1 increases transcriptionof other enzymes and proteins(green arrows)

Hypoxia (low pO2)

HIF-1

increasedlevelof HIF-1

Glucose

Glucosetransporter

Glycolyticenzymes

Pyruvate

ATP

ATP productionby glycolysisincreases

Acetyl-CoA

respiratory chainCitricacidcycle

ProteaseDegradesCOX4-1subunit

COX4-2subunit

ReplacesCOX4-1

NADH,FADH2

O2•O2

−, •OHElectron flow fromNADHand FADH2 toRespiratory chaindecreases

ROS production isreduced

Complex IV propertiesAre adapted to low pO2

lactatedehydrogenase

PDHkinase

pyruvatedehydrogenase(PDH)

O2

H2O

Complex IV

Lactate

Hypoxia-inducible factor (HIF-1)

Procaspase-9 monomers(inactive)

Caspase-9 dimers(active)

Cell deathProcaspase-3 Caspase-3

Procaspase-7 Caspase-7

These caspases lead to the death and re-sorption of the cell

Apoptosome causes dimerizationof procaspase-9, creating active caspase-9 dimers

Caspase-9 catalyzes proteo-lytic activation of caspase-3and caspase-7

Cytochrome c

DNAdamage

Developmentalsignal Stress ROS

Apaf-1 (apoptosisprotease activating factor-1)

Apoptosome

Cytochrome cmoves to cytosol

Permeability transitionpore complex opens

Binding of cytochrome cand ATP induces Apaf-1 toform an apoptosome

ATP

Role of cytochrome c in apoptosis

1. In mitochondria, hydride ions removed from substrates by NAD-linked dehydrogenasesdonate electrons to the respiratory (electron-transfer) chain, which transfers electrons to molecular O2 reducing it to H2O

2. The energy of electron flow is conserved by the concomitant pumping of protons across the membrane, producing an electrochemical gradient, the proton-motive force

3. Proton gradient provides the energy (in the form of the proton-motive force) for ATP synthesis from ADP and Pi by ATP synthase (FoF1 complex) in the inner membrane

4. ATP synthase carries out “rotational catalysis,” in which the flow of protons through Focauses each of three nucleotide-binding sites in F1 to cycle from (ADP + Pi)-bound to ATP-bound to empty conformations

5. Energy conserved in proton gradient can drive solute transport uphill across a membrane

6. In brown fat, electron transfer is uncoupled from ATP synthesis and the energy of fatty acid oxidation is dissipated as heat

7. Reactive oxygen species produced in mitochondria are inactivated by a set of protective enzymes that prevent oxidative stress

8. Mitochondrial cytochrome c, released into the cytosol can cause activation of caspasesand apoptosis