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Molecular Biochemistry I

Oxidative Phosphorylation

Copyright 1999-2007 by Joyce J. Diwan. All rights reserved.

Conventional view of mitochondrial structure is at right. Respiratory chain is in cristae of the inner membrane.
cristae

matrix intermembrane space

inner outer Spontaneous electron membrane mitochondrion membrane transfer through respiratory chain complexes I, III & IV is coupled to H+ ejection from the matrix to the intermembrane space. Because the outer membrane contains large channels, these protons may equilibrate with the cytosol. 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.

matrix intermembrane space outer membrane

cristae

inner membrane mitochondrion

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.

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 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.

Matrix

Spontaneous H+ + NADH NAD+ + 2H+ 2H+ + O2 H2O electron flow 2 e through each I Q III IV of complexes I, III, & IV is ++ + coupled to H cyt c 4H+ 4H+ 2H+ ejection from Intermembrane Space the matrix. 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.

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

2 eQ

III IV

++
4H
+

4H

cyt c

2H+

Intermembrane Space

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

Peripheral domain of a bacterial Complex I

NAD+ NADH matrix inner mitochondrial membrane membrane domain FMN peripheral domain
FMN

A
FMN

Complex I

membrane domain

N2 PDB 2FUG

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.

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

2 eQ

III IV

++
4H
+

4H

cyt c

2H+

Intermembrane Space

Complex III (bc1 complex):

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

O CH3O CH3 CH3 CH3O O (CH2 CH C

O-

e-

CH3O

CH3 CH3

CH2)nH

CH3O O

(CH2 CH

CH2)nH

coenzyme Q

coenzyme Q -

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

coenzyme QH2

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.

matrix Q

2 H+ Q.cyt bH
Complex III

QH2

QH2

cyt bL e-

e-

One version of Q Cycle:

Q 2 H+ intermembrane space

Fe-S

cyt c1 cyt c

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.

matrix

QH2 gives up 1eto the Rieske iron-sulfur center, Fe-S.

2 H+ Q.cyt bH
Complex III

QH2

QH2

Fe-S is reoxidized cyt bL eeby transfer of the - Q Q Fe-S e-to cyt c1, which + 2 H passes it out of the intermembrane space complex to 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.

cyt c1 cyt c

A 2nd is transferred from Q Q.QH2 QH2 the semiquinone to cyt bH cyt bL (heme bL) Complex III which passes it via cyt bH across the cyt b e L e membrane to - Q Q Fe-S cyt c1 another CoQ bound at a site on 2 H+ cyt c intermembrane space the matrix side. 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.

e-

matrix

2 H+

matrix Q

2 H+ Q.cyt bH
Complex III

QH2

QH2

cyt bL eQ

e-

Q 2 H+ intermembrane space

Fe-S

cyt c1 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.

matrix Q

2 H+ Q.cyt bH
Complex III

QH2

QH2

cyt bL eQ

e-

Q 2 H+ intermembrane space

Fe-S

cyt c1 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.

matrix

2 H+ Q.cyt bH QH2 QH2


Complex III

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

cyt bL eQ

e-

Q 2 H+ intermembrane space

Fe-S

cyt c1 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.

matrix Q

2 H+ Q.cyt bH
Complex III

QH2

QH2

cyt bL eQ

e-

Q 2 H+ intermembrane space

Fe-S

cyt c1 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-).

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

2 eQ

III IV

++
4H+ 4H+

cyt c

2H+

Intermembrane Space

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.

Complex III: Half of the homodimeric structure is shown. Approximate location of the membrane bilayer is indicated.

PDB 1BE3

Complex III (bc1 Complex)

membrane

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 bH heme bL

Fe-S heme c1

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.

PDB 1BE3

Complex III (bc1 Complex)

membrane

heme bH heme bL

After Fe-S extracts an e- from QH2, it moves closer to heme c1, to which it transfers the e-.
View an animation.

Fe-S heme c1

membrane

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 eacceptor, heme bL. This would help to prevent transfer of the 2nd electron from the semiquinone to Fe-S.

PDB 1BE3

Complex III (bc1 Complex)

heme bH heme bL

Fe-S heme c1

Complex III is an obligate homo-dimer.

PDB-1BGY

Complex III 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/grey heme c1 in the half of complex with proteins colored blue or green.

Fe-S heme c1

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

2 eQ

III IV

Complex IV (Cytochrome Oxidase):

++
4H+ 4H+

cyt c

2H+

Intermembrane Space

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.

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

2 eQ

III IV

++
4H+ 4H+

cyt c

2H+

Intermembrane Space

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.

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 sidechains 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 halfwire (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.

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

2 eQ

III IV

Simplified animation depicting:

++
4H+ 4H+

cyt c

2H+

Intermembrane Space

Ejection of a total of 20 H+ from the matrix per 4 etransferred 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.

ADP + Pi F1 Fo

ATP 3 H+

matrix

intermembrane space

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 F1 Fo

ATP 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+).

Matrix
H+ + NADH NAD+ + 2H+

ADP + Pi ATP 2H+ + O2 H2O

F1 Fo

2 eQ

III IV

++
4H+ 4H+

cyt c

2H+

3H+

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+

ADP + Pi ATP 2H+ + O2 H2O

F1 Fo

2 eQ

III IV

++
4H+ 4H+

cyt c

2H+

3H+

Intermembrane Space

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+

ADP + Pi ATP 2H+ + O2 H2O

F1 Fo

2 eQ

III IV

++
4H+ 4H+

cyt c

2H+

3H+

Intermembrane Space

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.

Transport of ATP, ADP, & Pi


ATP 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.

ADP + Pi ATP ATP4__ ++

matrix lower [H+]

3 H+

ATP4- ADP3- H2PO4- H+


higher [H+] cytosol

energy requiring reactions

ADP + 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 ATP4__ ++

matrix lower [H+]

Animation

3 H+

ATP4- ADP3- H2PO4- H+


higher [H+] cytosol

energy requiring reactions

ADP + Pi

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.

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

2 eQ

III IV

++
4H+ 4H+

cyt c

2H+

Intermembrane Space

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

For, summing up synthesis of ~P bonds via ox phos, assume:

2 eQ

III IV

++
4H+ 4H+

cyt c

2H+

Intermembrane Space

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).

All Quantities Per Glucose


Pathway Glycolysis Pathway Pyruvate Dehydrogenase Krebs Cycle Sum of Pathways NADH produced QH2 ~P bonds produced ATP or (via GTP direct FADH2) ~P bonds ~P bonds 1.5 or 2.5 1.5 per per NADH QH2 in in oxphos oxphos Total ~P bonds

An oxygen electrode may be used to record [O2] in a closed vessel. [O2] Electron transfer, e.g., NADH O2, is monitored by the rate of O2 disappearance.

ADP added

ADP all converted to ATP

time

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.

ADP added

[O2]

ADP all converted to ATP

time

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.

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. Reaction e- transfer (NADH O2) H+ ejection from matrix e- transfer with H+ ejection DG negative value* positive; depends on H+ gradient** algebraic 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

Matrix
H+ + NADH NAD+ + 2H+

ADP + Pi ATP 2H+ + O2 H2O

F1 Fo

2 eQ

III IV

++
4H
+

4H

cyt c

2H

3H+

Intermembrane Space

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+

ADP + Pi ATP 2H+ + O2 H2O

F1 Fo

2 eQ

III IV

++
4H+ 4H+

cyt c

2H+

3H+

Intermembrane Space

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.

OH NO2

NO2

2,4-dinitrophenol

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+.

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

2 eQ

III

IV

uncoupler
H+

4H

4H

cyt c

2H+

Intermembrane Space

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 eQ

III

IV

uncoupler
H+

4H

4H

cyt c

2H+

Intermembrane Space

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.

ADP + Pi F1
matrix

ATP 3 H+

Fo
intermembrane space

ATPase with H+ gradient dissipated

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.

Uncoupling Protein
An 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.