Oxidative PhosphorylationCopyright 1999-2007 by Joyce J. Diwan. All rights reserved.Molecular Biochemistry I

Respiration-linked H+ pumping out of the matrix conserves some of the free energy of spontaneous e- transfers as potential energy of an electrochemical H+ gradient.Conventional view of mitochondrial structure is at right.Respiratory chain is in cristae of the inner membrane. Spontaneous electron transfer throughrespiratory chain complexes I, III & IV is coupled to H+ ejectionfrom the matrix to the intermembrane space. Because the outer membrane contains large channels, these protons may equilibrate with the cytosol.

outer

membrane

inner

membrane

matrix

inter-

membrane

space

mitochondrion

cristae

3-D reconstructions based on electron micrographs of isolated mitochondria taken with a large depth of field, at different tilt angles have indicated that the infoldings of the inner mitochondrial membrane are variable in shape and are connected to the periphery and to each other by narrow tubular regions.

outer

membrane

inner

membrane

matrix

inter-

membrane

space

mitochondrion

cristae

between the lumen of cristae & the intermembrane space.There is evidence also that protons pumped out of the matrix spread along the anionic membrane surface and only slowly equilibrate with the surrounding bulk phase, maximizing the effective H+ gradient. Electron micrograph by Dr. C. Mannella of a Neurospora mitochondrion in a frozen sample in the absence of fixatives or stains that might alter appearance of internal structures. Wadsworth Center website. Tubular cristae connect to the inner membrane via narrow passageways that may limit the rate of H+ equilibration

A total of 10 H+ are ejected from the mitochondrial matrix per 2 e- transferred from NADH to oxygen via the respiratory chain. The H+/e- ratio for each respiratory chain complex will be discussed separately. Spontaneous electron flow through each of complexes I, III, & IV is coupled to H+ ejection from the matrix.

Matrix

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

2 e

I Q III IV

+ +

4H+ 4H+ 2H+

Intermembrane Space

cyt c

Complex I (NADH Dehydrogenase) transports 4H+ out of the mitochondrial matrix per 2e- transferred from NADH to CoQ.

Matrix

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

2 e

I Q III IV

+ +

4H+ 4H+ 2H+

Intermembrane Space

cyt c

Lack of high-resolution structural information for the membrane domain of complex I has hindered elucidation of the mechanism of H+ transport. Direct coupling of transmembrane H+ flux & e- transfer is unlikely, because the electron-tranferring prosthetic groups, FMN & Fe-S, are all in the peripheral domain of complex I.Thus is assumed that protein conformational changes are involved in H+ transport, as with an ion pump.

inner mitochondrial membrane

matrix

NAD+

NADH

Complex I

FMN

peripheral domain

membrane domain

( FMN

A B

( FMN

Peripheral domain of a bacterial Complex I

membrane domain

(

PDB 2FUG

( N2

Complex III (bc1 complex): H+ transport in complex III involves coenzyme Q (CoQ).

Matrix

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

2 e

I Q III IV

+ +

4H+ 4H+ 2H+

Intermembrane Space

cyt c

The Q cycle depends on mobility of coenzyme Q within the lipid bilayer.There is evidence for one-electron transfers, with an intermediate semiquinone radical.

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e + 2 H+

coenzyme Q

coenzyme QH2

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e

coenzyme Q

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Electrons enter complex III via coenzyme QH2, which binds at a site on the positive side of the inner mitochondrial membrane, adjacent to the intermembrane space. One version of Q Cycle:

2 H+

Q Q QH2 QH2

cyt bH

cyt bL

Q Q Fe-S cyt c1

2 H+

matrix

Complex III

e

intermembrane space

.

e

cyt c

The loss of one electron from QH2 would generate a semiquinone radical, shown here as Q-, though the semiquinone might initially retain a proton as QH. QH2 gives up 1e- to the Rieske iron-sulfur center, Fe-S.Fe-S is reoxidized by transfer of the e- to cyt c1,which passes it out of the complex to cyt c.

2 H+

Q Q QH2 QH2

cyt bH

cyt bL

Q Q Fe-S cyt c1

2 H+

matrix

Complex III

e

intermembrane space

.

e

cyt c

The fully oxidized CoQ, generated as the 2nd e- is passed to the b cytochromes, may then dissociate from its binding site adjacent to the intermembrane space.Accompanying the two-electron oxidation of bound QH2, 2H+ are released to the intermembrane space.A 2nd e- is transferred from the semiquinone to cyt bL (heme bL) which passes it via cyt bH across the membrane to another CoQ bound at a site on the matrix side.

2 H+

Q Q QH2 QH2

cyt bH

cyt bL

Q Q Fe-S cyt c1

2 H+

matrix

Complex III

e

intermembrane space

.

e

cyt c

In an alternative mechanism that has been proposed, the 2 e- transfers, from QH2 to Fe-S & cyt bL, may be essentially simultaneous, eliminating the semiquinone intermediate.

2 H+

Q Q QH2 QH2

cyt bH

cyt bL

Q Q Fe-S cyt c1

2 H+

matrix

Complex III

e

intermembrane space

.

e

cyt c

It takes 2 cycles for CoQ bound at the site hear the matrix to be reduced to QH2, as it accepts 2e- from the b hemes, and 2H+ are extracted from the matrix compartment. In 2 cycles, 2 QH2 enter the pathway & one is regenerated.

2 H+

Q Q QH2 QH2

cyt bH

cyt bL

Q Q Fe-S cyt c1

2 H+

matrix

Complex III

e

intermembrane space

.

e

cyt c

QH2 + 2H+(matrix) + 2 cyt c (Fe3+) Q + 4H+(outside) + 2 cyt c (Fe2+)Per 2e- transferred through the complex to cyt c, 4H+ are released to the intermembrane space. Animation

Overall reaction catalyzed by complex III, including net inputs & outputs of the Q cycle :

2 H+

Q Q QH2 QH2

cyt bH

cyt bL

Q Q Fe-S cyt c1

2 H+

matrix

Complex III

e

intermembrane space

.

e

cyt c

While 4H+ appear outside per net 2e- transferred in 2 cycles, only 2H+ are taken up on the matrix side.In complex IV, there is a similarly uncompensated proton uptake from the matrix side (4H+ per O2 or 2 per 2e-).

2 H+

Q Q QH2 QH2

cyt bH

cyt bL

Q Q Fe-S cyt c1

2 H+

matrix

Complex III

e

intermembrane space

.

e

cyt c

Thus there are 2H+ per 2e- that are effectively transported by a combination of complexes III & IV. They are listed with complex III in diagrams depicting H+/e- stoichiometry.

Matrix

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

2 e

I Q III IV

+ +

4H+ 4H+ 2H+

Intermembrane Space

cyt c

Complex III: Half of the homodimeric structure is shown. Approximate location of the membrane bilayer is indicated. Not shown are the CoQ binding sites near heme bH and near heme bL. The b hemes are positioned to provide a pathway for electrons across the membrane.

heme bL

heme c1

Fe-S

PDB

1BE3

Complex III (bc1 Complex)

membrane

heme bH

The domain with attached Rieske Fe-S has a flexible link to the rest of the complex. (Fe-S protein in green.)Fe-S changes position during e- transfer. After Fe-S extracts an e- from QH2, it moves closer to heme c1, to which it transfers the e-.View an animation.

heme bL

heme c1

Fe-S

PDB

1BE3

Complex III (bc1 Complex)

membrane

heme bH

After the 1st e- transfer from QH2 to Fe-S, the CoQ semiquinone is postulated to shift position within the Q-binding site, moving closer to its e- acceptor, heme bL. This would help to prevent transfer of the 2nd electron from the semiquinone to Fe-S.

heme bL

heme c1

Fe-S

PDB

1BE3

Complex III (bc1 Complex)

membrane

heme bH

Complex III is an obligate homo-dimer. Fe-S in one half of the dimer may interact with bound CoQ & heme c1 in the other half of the dimer.Arrows point at:Fe-S in the half of complex colored white/greyheme c1 in the half of complex with proteins colored blue or green.

heme c1

Fe-S

PDB-1BGY

Complex III homo-dimer

Electrons are donated to complex IV, one at a time, by cytochrome c, which binds from the intermembrane space.Each e- passes via CuA & heme a to the binuclear center, buried within the complex, that catalyzes O2 reduction: 4e- + 4H+ + O2 2H2O. Protons utilized in this reaction are taken up from the matrix compartment. Complex IV (Cytochrome Oxidase):

Matrix

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

2 e

I Q III IV

+ +

4H+ 4H+ 2H+

Intermembrane Space

cyt c

H+ pumping by complex IV:In addition to protons utilized in reduction of O2, there is electron transfer-linked transport of 2H+ per 2e- (4H+ per 4e-) from the matrix to the intermembrane space.

Matrix

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

2 e

I Q III IV

+ +

4H+ 4H+ 2H+

Intermembrane Space

cyt c

Structural & mutational studies indicate that protons pass through complex IV via chains of groups subject to protonation/deprotonation, called "proton wires." These consist mainly of chains of buried water molecules, along with amino acid side-chains, & propionate side-chains of hemes. Separate H+-conducting pathways link each side of the membrane to the buried binuclear center where O2 reduction takes place. These include 2 proton pathways, designated "D" & "K" (named after constituent Asp & Lys residues) extending from the mitochondrial matrix to near the binuclear center deep within complex IV.Images in web pages of: IBI, & Crofts.

A switch mechanism controlled by the reaction cycle is proposed to effect transfer of a proton from one half-wire (half-channel) to the other. There cannot be an open pathway for H+ completely through the membrane, or oxidative phosphorylation would be uncoupled. (Pumped protons would leak back.) Switching may involve conformational changes, and oxidation/reduction-linked changes in pKa of groups associated with the catalytic metal centers. Detailed mechanisms have been proposed.

Ejection of a total of 20 H+ from the matrix per 4 e- transferred from 2 NADH to O2 (10 H+ per O2). Not shown is OH- that would accumulate in the matrix as protons, generated by dissociation of water (H2O H+ + OH-), are pumped out. Also not depicted is the effect of buffering.Simplified animation depicting:

Matrix

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

2 e

I Q III IV

+ +

4H+ 4H+ 2H+

Intermembrane Space

cyt c

ATP synthase, embedded in cristae of the inner mitochondrial membrane, includes:F1 catalytic subunit, made of 5 polypeptides with stoichiometry a3b3gde.Fo complex of integral membrane proteins that mediates proton transport.

ADP + Pi ATP

F1

Fo

3 H+

matrix

intermembrane space

F1Fo couples ATP synthesis to H+ transport into the mitochondrial matrix. Transport of least 3 H+ per ATP is required, as estimated from comparison of: DG for ATP synthesis under cellular conditions (free energy required)DG for transfer of each H+ into the matrix, given the electrochemical H+ gradient (energy available per H+).

ADP + Pi ATP

F1

Fo

3 H+

matrix

intermembrane space

The Chemiosmotic Theory of oxidative phosphorylation, for which Peter Mitchell received the Nobel prize:Coupling of ATP synthesis to respiration is indirect, via a H+ electrochemical gradient.

Matrix

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

2 e

I Q III IV

+ +

4H+ 4H+ 2H+

Intermembrane Space

ADP + Pi ATP

3H+

F1

Fo

cyt c

Chemiosmotic theory - respiration: Spontaneous e- transfer through complexes I, III, & IV is coupled to non-spontaneous H+ ejection from the matrix. H+ ejection creates a membrane potential (DY, negative in matrix) and a pH gradient (DpH, alkaline in matrix).

Matrix

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

2 e

I Q III IV

+ +

4H+ 4H+ 2H+

Intermembrane Space

ADP + Pi ATP

3H+

F1

Fo

cyt c

Chemiosmotic theory - F1Fo ATP synthase: Non-spontaneous ATP synthesis is coupled to spontaneous H+ transport into the matrix. The pH & electrical gradients created by respiration are the driving force for H+ uptake. H+ return to the matrix via Fo "uses up" pH & electrical gradients.

Matrix

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

2 e

I Q III IV

+ +

4H+ 4H+ 2H+

Intermembrane Space

ADP + Pi ATP

3H+

F1

Fo

cyt c

Transport of ATP, ADP, & PiATP produced in the mitochondrial matrix must exit to the cytosol to be used by transport pumps, kinases, etc. ADP & Pi arising from ATP hydrolysis in the cytosol must reenter the matrix to be converted again to ATP.Two carrier proteins in the inner mitochondrial membrane are required. The outer membrane is considered not a permeability barrier. Large outer membrane VDAC channels are assumed to allow passage of adenine nucleotides and Pi.

Adenine nucleotide translocase (ADP/ATP carrier) is an antiporter that catalyzes exchange of ADP for ATP across the inner mitochondrial membrane. At cell pH, ATP has 4 (-) charges, ADP 3 (-) charges. ADP3-/ATP4- exchange is driven by, and uses up, membrane potential (one charge per ATP).

ADP + Pi ATP matrix

lower [H+]

_ _

3 H+ ATP4 ADP3 H2PO4 H+

higher [H+]

ADP + Pi cytosol

energy

requiring

reactions

ATP4

+ +

Phosphate re-enters the matrix with H+ by an electroneutral symport mechanism. Pi entry is driven by, & uses up, the pH gradient (equivalent to one mol H+ per mol ATP).Thus the equivalent of one mol H+ enters the matrix with ADP/ATP exchange & Pi uptake. Assuming 3H+ transported by F1Fo, 4H+ total enter the matrix per ATP synthesized. Animation

ADP + Pi ATP matrix

lower [H+]

_ _

3 H+ ATP4 ADP3 H2PO4 H+

higher [H+]

ADP + Pi cytosol

energy

requiring

reactions

ATP4

+ +

Questions: Based on the assumed number of H+ pumped out per site shown above, and assuming 4 H+ are transferred back to the matrix per ATP synthesized:What would be the predicted P/O ratio, the # of ATP synthesized per 2e- transferred from NADH to O2?What would be the predicted P/O ratio, if the e- source is succinate rather than NADH?

Matrix

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

2 e

I Q III IV

+ +

4H+ 4H+ 2H+

Intermembrane Space

cyt c

2.5 ~P bonds synthesized during oxidation of NADH produced via Pyruvate Dehydrogenase & Krebs Cycle (10 H+ pumped; 4 H+ used up per ATP).1.5 ~P bonds synthesized per NADH produced in the cytosol in Glycolysis (electron transfer via FAD to CoQ).1.5 ~P bonds synthesized during oxidation of QH2 produced in Krebs Cycle (Succinate Dehydrogenase electrons transferred via FAD & Fe-S to coenzyme Q).For, summing up synthesis of ~P bonds via ox phos, assume:

Matrix

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

2 e

I Q III IV

+ +

4H+ 4H+ 2H+

Intermembrane Space

cyt c

All Quantities Per Glucose

Pathway

NADH produced

QH2 produced(via FADH2)

~P bonds ATP or GTP direct

~P bonds1.5 or 2.5 per NADH in oxphos

~P bonds1.5 per QH2 in oxphos

Total ~P bonds

Glycolysis Pathway

Pyruvate Dehydrogenase

Krebs Cycle

Sum of Pathways

Above is represented an O2 electrode recording while mitochondria respire in the presence of Pi and an e- donor (succinate or a substrate of a reaction to generate NADH). The dependence of respiration rate on availability of ADP, the ATP Synthase substrate, is called respiratory control.An oxygen electrode may be used to record [O2] in a closed vessel.Electron transfer, e.g., NADH O2, is monitored by the rate of O2 disappearance.

[O2]

time

ADP added

ADP all converted to ATP

a

b

c

Respiratory control ratio is the ratio of slopes after and before ADP addition (b/a).P/O ratio is the moles of ADP divided by the moles of O consumed (based on c) while phosphorylating the ADP.

[O2]

time

ADP added

ADP all converted to ATP

a

b

c

Chemiosmotic explanation of respiratory control: Electron transfer is obligatorily coupled to H+ ejection from the matrix. Whether this coupled reaction is spontaneous depends on pH and electrical gradients.ReactionDGe- transfer (NADH O2)negative value*H+ ejection from matrixpositive; depends on H+ gradient**e- transfer with H+ ejectionalgebraic sum of above*DGo' = -nFDEo' = -218 kJ/mol for 2 e- NADH O2. **For ejection of 1 H+ from the matrix: DG = RT ln ([H+]cytosol/[H+]matrix) + FDY DG = 2.3 RT (pHmatrix - pHcytosol) + FDY

With no ADP, H+ cannot flow through Fo. DpH & DY are maximal. As respiration/H+ pumping proceed, DG for H+ ejection increases, approaching that for e- transfer. When the coupled reaction is non-spontaneous, respiration stops. This is referred to as a static head. In fact there is usually a low rate of respiration in the absence of ADP, attributed to H+ leaks.

Matrix

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

2 e

I Q III IV

+ +

4H+ 4H+ 2H+

Intermembrane Space

ADP + Pi ATP

3H+

F1

Fo

cyt c

When ADP is added, H+ enters the matrix via Fo, as ATP is synthesized. This reduces DpH & DY. DG of H+ ejection decreases. The coupled reaction of electron transfer with H+ ejection becomes spontaneous. Respiration resumes or is stimulated.

Matrix

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

2 e

I Q III IV

+ +

4H+ 4H+ 2H+

Intermembrane Space

ADP + Pi ATP

3H+

F1

Fo

cyt c

Uncoupling reagents (uncouplers) are lipid-soluble weak acids. E.g., H+ can dissociate from the OH group of the uncoupler dinitrophenol. Uncouplers dissolve in the membrane and function as carriers for H+.

2,4-dinitrophenol

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Uncouplers block oxidative phosphorylation by dissipating the H+ electrochemical gradient. Protons pumped out leak back into the mitochondrial matrix, preventing development of DpH or DY.

Matrix

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

2 e

I Q III IV

4H+ 4H+ 2H+ H+

Intermembrane Space

uncoupler

cyt c

With uncoupler present, there is no DpH or DY. DG for H+ ejection is zero DG for e- transfer coupled to H+ ejection is maximal (spontaneous). Respiration proceeds in the presence of an uncoupler, whether or not ADP is present.

Matrix

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

2 e

I Q III IV

4H+ 4H+ 2H+ H+

Intermembrane Space

uncoupler

cyt c

DG for H+ flux is zero in the absence of a H+ gradient. Hydrolysis of ATP is spontaneous.The ATP Synthase reaction runs backward in presence of an uncoupler.

ADP + Pi ATP

F1

Fo

3 H+

ATPase with H+ gradient dissipated

matrix

intermembrane space

Uncoupling ProteinAn uncoupling protein (thermogenin) is produced in brown adipose tissue of newborn mammals and hibernating mammals. This protein of the inner mitochondrial membrane functions as a H+carrier. The uncoupling protein blocks development of a H+ electrochemical gradient, thereby stimulating respiration. DG of respiration is dissipated as heat. This "non-shivering thermogenesis" is costly in terms of respiratory energy unavailable for ATP synthesis, but provides valuable warming of the organism.