Oxidative phosphorylation

Oxidative phosphorylation in eukaryotes occurs exclusively in their mitochondria.

Mitochondria are small endosymbiotic organelles in eukaryotes with a complex double-membrane system.

As previously discussed, mitochondria convert pyruvate into carbon dioxide and water via the Krebs cycle. This produces NADH and a little ATP. The NADH, which would otherwise accumulate until there was no NAD left, is re-oxidised by the oxidative phosphorylation respiratory chain to regenerate the NAD, and (as a fabulous bonus), generate loads of ATP too, because this oxidation is coupled to the production of a proton gradient across their inner membrane, and these protons flow down their gradient via F_OF₁-ATPase (ATP synthase), making ATP.

Mitochondria are believed to be the product of an endosymbiosis 2500 MYA. They probably originated from an intracellular proteobacterial parasite of proto-eukaryotic cells (something like Bdellovibrio). Chloroplasts derived from a similar symbiosis with cyanobacteria (closely related to Prochloron).

Mitochondria and chloroplasts were once free living proteobacteria and cyanobacteria.

Since oxidative phosphorylation is much more efficient (30 vs. 2 ATP per glucose) than anaerobic respiration, and photosynthesis allows growth in the absence of exogenous carbon and reducing agents, what may have begun as a parasitic or predatory relationship between the Ur-eukaryote and its bacterial passengers, developed into a mutualism. The mitochondrial symbiosis were probably a one-off, but chloroplasts may not have been. Dinoflagellates even have secondary endosymbionts (like Russian matryoshka dolls): a chloroplast within an alga within another alga. There is a great deal of evidence for endosymbiosis these days (the theory was once considered very unlikely):

Inner membrane = Remains of bacterial plasmalemma.
Outer membrane = Remains of food vacuole.
Cristae = Like mesosome infoldings.
Circular DNA lacking histones in matrix = Remains of bacterial chromosome.
Small ribosomes = Like bacterial 70S ribosomes (eukaryotes have 80S).

The symbiosis has gone far beyond a simple ingestion. The human mtDNA (mitochondrial DNA) genome contains just 37 genes. These are mostly tRNAs, with some of the proteins of oxidative phosphorylation:

7/27 of Complex I
0/4 of Complex II
1/9 of Complex III
3/13 of Complex IV
2/12 of Complex V

The rest is now nuclear encoded and imported via the TOM/TIM transport system, showing that over evolutionary time, the genes of the mitochondria have either been lost, or (when essential) transferred to the nuclear genome.

Mitochondrion showing functions of its various structures.

The job of mitochondria is to convert pyruvate to ATP and carbon dioxide. This is achieved by the interaction of NADH, and one Krebs cycle intermediate (succinate) with the inner mitochondrial membrane. This membrane contains five huge protein complexes, which serve to remove electrons from NADH, regenerating NAD, and in-so-doing, to generate a proton gradient across the membrane than may be used to drive ATP synthesis.

The electron transport chain consist of four complexes, plus an ATP synthase.

The five complexes are named I, II, III, IV and V. We shall discuss them in turn:

NADH is oxidised to NAD, producing UQH2 from UQ.
Electrons from the oxidation of NADH pass through flavin, FeS and UQ centres before being dumped onto ubiquinone. This pumps four protons, and attaches two others to UQ.

Complex I.

Complex I is NADH dehydrogenase. It removes two electron from NADH, and transfers them to ubiquinone in the mitochondrial membrane. As the two electrons pass through various flavin (FMN), iron-sulfur (FeS) and quinone (UQ) centres, four protons are pumped across complex I into the inter-membrane space (per NADH). When the electrons are deposited onto UQ (ubiquinone), the UQ takes up a further two protons from the matrix side, to form ubiquinol (UQH₂) (these are excluded from the pump-count for this complex). It produces 1 UQH₂, per NADH oxidised. The ultimate source of the NADH and the electrons is the oxidation of ketoglutarate, malate etc in the Krebs cycle.

The ubiquinol formed feeds into a UQ 'pool' inside the membrane, and diffuses to complex III.

Complex II.

Complex II is also called succinate dehydrogenase, and is the only membrane bound enzyme of the Krebs cycle. The dehydrogenation of succinate has too small a ∆G for any H⁺ pumping, so this complex only generates 1 UQH₂ per succinate oxidised, and pumps no protons. It gets its electrons from the oxidation of succinate only, and feeds them via flavin (FAD) and an iron-sulfur cluster into the UQH₂ pool.

Succinate dehydrogenase faces the matrix space, and does not span the membrane, so no protons are pumped.
Succinate is oxidised to generate fumarate, and UQH2 from UQ.

UQ is reduced to UQH2 via a reactive free radical called a semiquinone.

Ubiquinone and ubiquinol

Ubiquinone (UQ) ferries electrons from complexes I and II to complex III. Note the long hydrophobic chain: UQ/UQH₂ can migrate actually dissolved within the membrane.

Partial reduction of UQ generates ubisemiquinone radicals (UQH·), which are very dangerous and must be rapidly reduced to UQH₂.

Complexes I and II both feed into a pool of ubiquinol (UQH₂) actually inside the inner mitochondrial membrane (dissolved in the fatty acid tails).

The reason semiubiquinone is dangerous is that is can generate superoxide radicals, which are hugely oxidising free-radicals.

UQH· + O₂ → UQ + H⁺ + O₂⁻·

Superoxide will dismutate to hydrogen peroxide.

2O₂⁻· + 2H⁺ → O₂ + H₂O₂

Hydrogen peroxide will undergo Fenton reaction with haem iron to produce hydroxyl radicals which are lethally destructive.

Fe²⁺ + H₂O₂ → Fe³⁺ + OH⁻ + OH·

Mitochondria therefore contain superoxide dismutase and glutathione (GSH) peroxidase to cope with these agents of oxidative stress.

2GSH + H₂O₂ → GSSG + 2H₂O

UQH2 is oxidised to UQ, and the electrons used to reduce two molecules of cytochrome-c.

Complex III.

This is also called cytochrome reductase (or oxidoreductase). It pumps 4 H⁺ per UQH₂ (including the two attached by complex I or II to UQ), and produces 2 cyt-c_RED (reduced cytochrome-c) per UQH₂ oxidised. The iron in the haem groups of b and c cytochromes goes from Fe³⁺ to Fe²⁺. The complex manages to pump 4 protons by running a nasty bit of biochemistry called the Q-cycle, which delivers the two electrons from one UQH₂ to two cyt-c molecules, which only carry one electron each.

The 'Q-cycle' is a preposterously complicated way of transferring electrons from the two-electron-carrying UQH₂ to the single-electron carrying cytochrome-c (cyt-c). A UQH₂ gives up its protons to the IMS. One of its electrons is carried through FeS and cyt-c₁ to the mobile cytochrome-c. The second of its electrons is carried through two cyt-b centres and is dumped back onto another UQ molecule to form a semiquinone radical. The same process then happens again with a further UQH₂, fully reducing the semiquinone to UQH₂. Note that the whole process consumes two UQH₂, but generates one back, so there is a net oxidation of just one UQH₂.

The Q-cycle recycles one electron given up by each UQH2 oxidation back onto another UQ molecule. This doubles the number of protons pumped by the quinone system.
One electron from UQH2 is used to reduce cyt-c, the other is used to half-reduce a UQ in the membrane to semiquinone. This is accompanied by the release of two protons per UQH2 into the IMS. One electron from a second UQH2 is used to reduce cyt-c, and the other is used to regenerate a UQH2 in the membrane from the semiquinone radical produced earlier, with uptake of protons from the matrix. This is again accompanied by the release of two protons per UQH2 into the IMS. In upshot, only one net UQH2 has been oxidised, but four protons have been pumped.

Cytochrome-b.

Cytochrome b and c

Cytochromes are small proteins containing a haem group (much like myoglobin or haemoglobin). They are grouped into three types (a, b and c) according to the type of haem and how it is bound into the protein.

Cytochrome-b proteins contain an iron protoporphyrin-IX prosthetic group, which is bound by dipole interactions. The Fe ion is hexacoordinated: 4 ligands from the N's of haem, and 2 from the histidines in the protein.

Cytochrome-b showing coordination to protein.

Cytochrome-c.

Cytochrome-c contains a haem-c prosthetic group bound covalently by its ring to cysteines in the protein. The Fe ion is hexacoordinated: 4 from the N's of haem, 1 from a histidine in the protein, and 1 from methionine in the protein.

Cytochrome-c showing coordination to protein.

Two cytochrome-c molecules are used to oxidise half a molecule of oxygen to water.
Cytochrome oxidase dumps electrons from cytochrome-c onto oxygen, generating water, and pumping one proton per cytochrome-c.

Complex IV.

Complex IV is more commonly termed cytochrome oxidase (or even just cyt-ox). It pumps 2 H⁺ per 2 cyt-c_RED, and produces 1 H₂O per 2 cyt-c_RED oxidised. Complex IV receives its electrons from cytochrome-c, which is a small, mobile protein that diffuses from complex III to complex IV. The electrons are passed through a number of cytochrome-a and copper ion centres. Cu_B and cyt-a₃ actually perform the reduction of oxygen to water. Each NADH originally oxidised yields 2 electrons, and these are enough to reduce half an O₂ molecule to H₂O (i.e. four electrons - two NADH - are required to reduce a whole molecule of dioxygen).

Iron and copper ions bind oxygen and reduce it in stages.

Cytochrome-a contains a haem-a prosthetic group bound by 'hydrophobic forces' to the protein. It also has a long phytol tail (just like chlorophyll). The Fe ion is pentacoordinated: 4 from the N's of haem, 1 from a histidine in the protein. This leaves a binding site for oxygen.

Cytochrome-a showing coordination to protein.

The reason that electrons flow through the various complexes is that earlier stages have lower redox potentials, so can provide electrons for downstream reactions.

Redox potentials of components of the respiratory chain.

ATPase rotates in the membrane during ATP synthesis.
The rotation of the ATPase can be observed by attaching a fluorescent actin tail to the γ subunit of an isolated F₁ 'headpiece'. As the F₁ hydrolyses ATP, γ is forced to rotate (anticlockwise as viewed from the [absent] F_O).

Complex V.

Complex V is ATP synthase (an F-type ATPase). It converts an H⁺ gradient into ATP, producing c. 1 ATP per 3 or 4 H⁺ (the stoichiometry is still not quite certain, and may well vary between different organelles and organisms). The ATPase actually acts as (probably Brownian) motor: the F_O subunit rotates as protons flow through and ATP is synthesised due to the conformational changes this causes in F₁. It probably requires 3 protons to actually form one molecule of ATP, but one further proton is required to translocate ATP out of (and ADP/phosphate into) the matrix.

One ATP is generated per 4 protons allowed to flow back across the membrane.

As protons flow through the a/b subunits (the stator) of F_O, they force the ring of twelve c subunits (the rotor) in the membrane to rotate. This rotation is transmitted to the γ/ε subunits (the stalk) of F₁, which change the conformation of the α/β subunits (the headpiece) of F₁, making ADP and phosphate react to form ATP inside the β subunits. The headpiece is prevented from rotating by the binding of δ to the a/b stator, which is itself firmly anchored in the membrane.

Upshot

The electron transport chain carries the electrons produced by the oxidation of NADH to NAD through complexes I, III and IV. This electron transport is used to drive proton pumping through the membrane. The electrons are eventually dumped onto oxygen, which is reduced to water. The proton gradient built up by these processes is used to drive the F_OF₁ ATPase (in reverse) to generate ATP. The oxidation of 1 NADH pumps (about) 10 protons. ATPase generates (about) 1 ATP from 4 protons.

Source	Anaerobic	Aerobic
Glycolysis	2 ATP (substrate level phosphorylation)	2 ATP (substrate level phosphorylation)
Glycolysis	2 NADH → 0 ATP (cytosol)	2 NADH → 5 ATP
Krebs	-	2 ATP/GTP (substrate level phosphorylation)
	-	8 NADH → 20 ATP
	-	2 FADH₂ (succinate) → 3 ATP
Approximate total yield	2 ATP	32 ATP

Take the energy budgets with a pinch of salt.

NADH must be translocated into the mitochondrion from the cytoplasm: this costs ATP.
The complexes are not 100% efficient.
The inner membrane is not 100% impermeable.
There is still argument about the stoichiometry of the various complexes.

Aerobic respiration is approximately 15 times more efficient than anaerobic. The P/O ratio (ATP made per oxygen atom reduced) is about 3 for NADH and 2 for succinate (FADH₂). In books, you will find many different estimates of the ATP to glucose ratio, the number of protons pumped by each complex, the proton to ATP ratio for ATPase, etc. The numbers presented here are not to be taken as the definitive version!

Chemiosmosis

Chemiosmosis is the name given to the generation of ATP from a proton gradient. It occurs in all living things:

Chemiosmosis generates ATP from proton gradients.

Photosynthetic archaea

In some Archaea, the proton gradient is generated by light.

Purple proteobacteria

In some proteobacteria, the proton gradient is generated by light.

Mitochondria

In mitochondria, the proton gradient is generated using NADH.

Chloroplasts

In plastids, the proton gradient is generated by light.

The components of the chemiosmotic systems are similar too:

Mitochondria, chloroplasts and purple-bacteria use a cytochrome complex to run the Q-cycle on quinones.
The prosthetic group of cytochromes and chlorophyll are both porphyrins with phytanol tails.
Many of the proteins also contain FeS and quinone clusters.
Chloroplasts transfer electrons from water to NADPH, mitochondria transfer electrons from NADH to oxygen.
All use an F-type ATPase in reverse to generate ATP.

Chemiosmosis can be disrupted by a variety of chemicals. In oxidative phosphorylation, some of these inhibitors are quite infamous:

Respiratory chain inhibitors.

Rotenone blocks electron flow from FeS to UQ in complex I.
Antimycin-a blocks electron flow from cyt-b_L to UQ in complex III (Q-cycle).
Cyanide and carbon monoxide block access of O₂ to cyt-a₃ in complex IV.
Oligomycin damages F_O subunit of complex V.

These inhibitors were useful in the early research on the respiratory chain, and are still used to halt the chain at a particular point to study the stoichiometry of proton pumping.

Chemiosmosis works by generating a proton-motive force. The proton-motive force is the free energy associated with a gradient of protons across a proton-impermeable membrane. It is composed of two components: a chemical concentration gradient and an electrochemical charge gradient.

∆G = R T ln ( [H⁺]_matrix ⁄ [H⁺]_ims) − z F ∆E_m

The proton motive force has two components: an electrical one, and a concentration (pH) one.

As well as simple inhibition, we can also uncouple electron transport from ATP synthesis by destroying the proton motive force. This is called uncoupling. Ionophores are rather good at this.

Dinitrophenol (DNP) is a proton ionophore (a weak acid). It carries protons across a membrane in a similar way to valinomycin with potassium ions. Pentachlorophenol (PCP) acts in a similar way to DNP. It was widely used as a biocide, especially in pallet board manufacture as a fungicide, but is now banned by the Biocidal Products Directive, because of its extreme toxicity and environmental persistence.

DNP

PCP

Other ionophores are more specific. Valinomycin is a potassium ionophore: it destroys ∆E_m but not ∆pH: It uncouples ATP synthesis in mitochondria but not in chloroplasts, indicating that mitochondria use ∆E_m (−150 mV), but not ∆pH (usually only about 0.5 pH units). Nigericin is an antiport ionophore that swaps H⁺ for K⁺. This is charge-neutral, so destroys ∆pH but has no effect on ∆E_m. Nigericin effectively uncouples chloroplast ATP synthesis, but not mitochondrial, indicating that chloroplasts use ∆pH (usually about 4 units: stroma at pH 8, lumen at pH 4), but not ∆E_m (0 mV).

Sometimes, organisms want to generate heat rather than ATP from chemiosmotic gradients. Brown fat tissue is mitochondria-rich adipose tissue. Lipid is oxidised and a proton gradient built up, but this is uncoupled from ATP synthesis by thermogenin. Thermogenin is a proton channel found in brown fat mitochondria. The flow of protons through the membrane generates heat.

Thermogenin destroys the electrochemical gradient generating heat.

Plants have a number of interesting 'extras' in their mitochondrial membranes. They have an intermembrane-space side NADPH dehydrogenase and a matrix-side rotenone-insensitive NADH dehydrogenase. They also have an alternative oxidase (alt-ox) that uncouples electron transport from ATP synthesis. This system (in theory) could completely uncouple NADH oxidation from ATP production, generating almost nothing but heat from the Krebs cycle.

The alternative oxidase generates heat but no ATP from NAD(P)H oxidation.

What is the alternative oxidase for? It is known to be under hormonal control; it is stimulated by the plant hormone salicylic acid (Aspirin). It produces heat and could allows Krebs cycle (and the associated amino-acid pathways) to run even if ATP is not required by the cell (the energy overspill hypothesis). It also removes oxygen and prevents the build-up of reactive oxygen species produced by respiration and photosynthesis. However, in most cases, no-one really knows what it is 'for'. However, in aroids (arum lilies) it is know that the alternative oxidase generates heat in their inflorescence, volatilising amines and other fly-attracting chemicals. Skunk cabbage uses the heat generated to escape snow burial of its flowers.

Diagrams

You may find this diagram useful:

Krebs cycle.

Test yourself

Why does the oxidation of succinate produce less ATP than that of malate?
Why do mitochondria contain glutathione?

Complete the table:

	Archaea	Purple bacteria	Chloroplasts	Mitochondria
Energy source
Pump
Products

Which intermediates would you expect to accumulate if you treated mitochondria with antimycin-A?
Valinomycin uncouples electron transport from ATP synthesis, but it is not a proton ionophore. How does it work?

Answers

Bibliography

Berg, J. M., Tymoczko, J. L. and Stryer, L. (2006). Biochemistry. 6th edition. W. H. Freeman and Company, New York. 502-540. "Oxidative phosphorylation"
Grigorieff, N. (1999). Structure of the respiratory NADH:ubiquinone oxidoreductase (complex I). Current Opinion in Structural Biology 9:476-483. http://dx.doi.org/10.1016/S0959-440X(99)80067-0
Mitchell, P. (1976). Keilin's respiratory chain concept and its chemiosmotic consequences. Science 206:1148-1159
Noji, H., et al. (1997). Direct observation of the rotation of F₁-ATPase. Nature 386:299-302. http://dx.doi.org/doi:10.1038/386299a0
Saraste, M. (1999). Oxidative phosphorylation at the fin de siecle. Science 283:1488-1493. http://dx.doi.org/10.1126/science.283.5407.1488
Shultz, B. E. and Chan, S. I. (2001). Structures and proton-pumping strategies of mitochondrial respiratory enzymes. Annual Reviews of Biophysics and Biomolecular Structure 30:23-65. http://dx.doi.org/doi:10.1146/annurev.biophys.30.1.23

This page has been peer reviewed by 3 people. Thanks to Henry Mori and Jeannette Doeller for their corrections.

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