Molecular Biochemistry I

Oxidative Phosphorylation: Chemiosmotic Coupling

Contents of this page:
Mitochondrial structure
H+ ejection from the mitochondrial matrix linked to respiration
Complex IComplex III (Q cycle)Complex IV
Chemiosmotic theory of oxidative phosphorylation
Transport of ADP/ATP & Pi - role of DpH & DY
Respiratory control & uncoupling

A conventional view of mitochondrial structure is represented at right. The respiratory chain is embedded in cristae of the inner membrane.

Spontaneous electron 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, protons in the intermembrane space may equilibrate with the cytosol.

Respiration-linked pumping of protons out of the mitochondrial matrix conserves some of the free energy of spontaneous electron transfers as potential energy of an electrochemical H+ gradient.

3-D reconstructions based on electron micrographs taken with 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. 

At right is an electron micrograph, provided by Dr. Carmen Mannella of the Wadsworth Center, of a Neurospora mitochondrion in a frozen sample in the absence of fixatives or stains that might alter the appearance of internal structures.

Tubular cristae connect to the inner membrane via narrow passageways that may limit the rate of H+ equilibration between the lumen of cristae and the intermembrane space. (See Fig 22-4 in Voet & Voet, Biochemistry, 3rd Edition, p. 799, and a Wadsworth Center website.) 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.

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A total of 10 protons are ejected from the mitochondrial matrix per 2 electrons transferred from NADH to oxygen via the respiratory chain. The H+/e- ratio for each respiratory chain complex will be discussed separately. (See also article by P. Hinkle).

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

Lack of high-resolution structural information for the membrane domain of complex I has hindered elucidation of the mechanism of H+ transport through this complex. Direct coupling of transmembrane proton flux and electron transfer is unlikely, because the electron-transferring prosthetic groups, FMN and iron-sulfur centers, are all located in the peripheral domain of complex I (see notes on electron transfer chain). Thus it is assumed that protein conformational changes are involved in H+ transport, as with an ion pump.

Complex III (bc1 complex): H+ transport in complex III involves coenzyme Q (CoQ). The "Q cycle" depends on the mobility of CoQ within the lipid bilayer. There is evidence for one-electron transfers, with an intermediate semiquinone radical state.

Q Cycle:

  • 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.
  • QH2 gives up one electron to the Rieske iron-sulfur center (Fe-S).
    Fe-S is reoxidized by transfer of the electron to cytochrome c1, which passes it out of the complex to cytochrome c
    The loss of one electron from QH2 would generate a semiquinone radical, shown here as Q-, although the semiquinone might initially retain a proton as QH.
  • A second electron is transferred from the semiquinone to cytochrome bL (heme bL), which passes it across the membrane via cytochrome bH (heme bH) to another CoQ bound at a site on the matrix side of the membrane.
    The fully oxidized CoQ, generated as the second electron 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.

Q cycle (one version)

In an alternative mechanism that has been proposed, the two electron transfers from QH2 to Fe-S & cyt bL may be essentially simultaneous, eliminating the semiquinone intermediate. (See the list of recent reviews for more detailed discussions of proposed mechanisms.)

It takes 2 cycles for CoQ, bound at the site near the matrix side of the membrane, to be reduced to QH2, as it accepts 2 electrons from the b hemes and 2 H+ are extracted from the matrix compartment. In 2 cycles, 2 QH2 enter the pathway, and one is regenerated.

Overall reaction catalyzed by complex III, including net inputs and outputs of the Q cycle
QH2 + 2H+(matrix side) + 2 cyt c (Fe3+) Q + 4H+(outside) + 2 cyt c (Fe2+)

Per 2e- transferred through the complex to cytochrome c, 4H+ are released to the intermembrane space. While 4H+ appear outside per net 2e- transferred in 2 cycles, only 2H+ are taken up on the matrix side. In respiratory chain complex IV (see below), there is a similarly uncompensated uptake of protons from the matrix side (4H+ per O2 or 2 per 2e-). 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 (e.g., see above) depicting H+/e- stoichiometry.

of e- & H+ transfer in Complex III 

Half of the homo-dimeric complex III is depicted at right. The approximate location of the membrane bilayer is indicated. Not shown are the CoQ binding sites near heme bH near heme bL.

The b hemes are positioned to provide a pathway for electron transfer across the membrane.

The protein domain with attached Rieske iron-sulfur center (labeled Fe-S) has a flexible link to the rest of the complex. At right, the iron-sulfur center protein is colored green. The iron-sulfur center changes position during electron transfer. After Fe-S extracts an e- from QH2, it moves closer to heme c1 (cytochrome c1) to which it transfers the e-. View an animation of this domain movement by the Crofts lab.

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

Complex III is an obligate homo-dimer. The iron-sulfur center in one half of the dimer may interact with bound CoQ and heme c1 in the other half of the dimer.

At right, arrows indicate the positions of:

  • Fe-S in the half of the complex whose proteins are colored white/grey
  • heme c1 in the half of the complex whose proteins are colored in shades of blue or green.

Crystal structures on which these diagrams are based, (PDB 1BE3 & 1BGY) were solved by S. Iwata et al, in 1998.

Complex IV (Cytochrome Oxidase): As discussed in the section on the respiratory chain, electrons are donated to complex IV, one at a time, by cytochrome c, which binds from the intermembrane space. Each electron passes via CuA and heme a to the binuclear center, buried within the complex, that catalyzes oxygen reduction:
    4e- + 4H+ + O2 2H2O.
rotons utilized in this reaction are taken up from the matrix compartment.

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

Structural and 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, and propionate side-chains of the hemes.

Separate H+-conducting pathways link each side of the membrane to the buried binuclear center where O2 reduction takes place. These include two proton pathways, designated "D" and "K" (named after constituent Asp and Lys residues) extending from the mitochondrial matrix to near the binuclear center deep within complex IV.
See diagram p. 826, and images in:
   a webpage maintained by the Institute of Biological Information Processing in Germany,
   a webpage maintained by A. 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; see below). The process of 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 (see articles on oxidase).

A simplified animation of the entire respiratory chain, at right, includes a simplified representation of the Q cycle. A total of 20 H+ is shown being transported out of the matrix, per 4 e- transferred from 2 NADH to O2 (10 H+ per O2). 

Not shown are OH- ions 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 on the pH gradient of buffering. 

of the Respiratory Chain 

The ATP synthase, which is embedded in cristae of the inner mitochondrial membrane, includes the following major subunits:

  • F1 - the catalytic subunit, made of 5 polypeptides with stoichiometry a3b3gde.

  • Fo - a complex of integral membrane proteins that mediates proton transport. 

The F1Fo complex couples ATP synthesis to H+ transport into the mitochondrial matrix. Transport of at least 3H+ per ATP synthesized is required, as estimated from a comparison of the following (calculated in the Tutorial):

ATP Synthase structure and mechanism are discussed in more detail elsewhere.

The Chemiosmotic Theory of oxidative phosphorylation, for which Peter Mitchell received the Nobel prize is summarized in the diagram at right.

The Chemiosmotic Theory states that coupling of electron transfer to ATP synthesis is indirect, via a H+ electrochemical gradient:

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

  2. F1Fo ATP Synthase: Non-spontaneous ATP synthesis is coupled to spontaneous H+ transport into the matrix compartment. The pH and electrical gradients created by respiration are together the driving force for H+ uptake.
    Return of protons to the matrix via Fo "uses up" the pH and electrical gradients.

ATP produced in the mitochondria must exit to the cytosol to be used by transport pumps, kinases, etc. ADP and Pi, arising from ATP hydrolysis in the cytosol, must re-enter the mitochondria to be converted again to ATP. 

Two carrier proteins in the inner mitochondrial membrane are required for this metabolic cycle. The outer membrane is considered to be not a permeability barrier. The large VDAC channels in the outer membrane are assumed to allow passage of adenine nucleotides and Pi

  1. The Adenine Nucleotide Translocase (ADP/ATP carrier) is an antiporter that catalyzes exchange of ADP for ATP across the inner mitochondrial membrane (p. 496). At cellular pH, ATP has four negative charges, while ADP has 3 negative charges. ADP3-/ATP4- exchange is driven by, and uses up, the membrane potential generated by respiration (one charge per ATP).

  2. Phosphate reenters the mitochondrial matrix with H+, by an electroneutral symport mechanism. Pi entry is driven by and uses up the pH gradient (equivalent to one mole of H+ per mole of ATP).

Thus the equivalent of one mol of H+ enters the matrix with ADP/ATP exchange and Pi uptake. Assuming transport of 3 mol H+ by F1Fo, a total of 4H+ would enter the mitochondrial matrix per ATP synthesized.

At right is an animation depicting functioning of the adenine nucleotide translocase and phosphate symport, along with the ATP synthase.

of ADP/ATP & Pi Translocation 

The phenomenon of respiratory control is the subject of today's studio exercise. An oxygen electrode may be used to record [O2] in a closed vessel (diagram p. 804). Electron transfer, e.g., from NADH to O2, is monitored by recording the rate of disappearance of O2.

At right is an idealized representation of an oxygen electrode recording while mitochondria respire in the presence of Pi, along with an electron donor (e.g., succinate, or a substrate of a reaction that will generate NADH).

The dependence of respiration rate on availability of ADP, the substrate for the ATP Synthase, is called respiratory control.

The respiratory control ratio is the ratio of slopes after and before ADP addition (b/a).

The P/O ratio is the moles of ADP added, divided by the moles of O consumed (based on c) while phosphorylating the added ADP.

Studio exercise involving calculation of P/O ratio and respiratory control ratio.

Chemiosmotic explanation of respiratory control: 

Electron transfer is obligatorily coupled to H+ ejection from the matrix. Whether this coupled reaction is spontaneous depends on the pH and electrical gradients.


Free energy change

e- transfer (e.g., NADH to O2)

a negative value*

H+ ejection from the matrix

a positive value that varies with the H+ gradient**

e- transfer coupled to H+ ejection

algebraic sum of the above

*DGo' = - nFDEo' = -218 kJ/mol, for transfer of 2 e- from NADH to O2.

** For ejection of one H+ from the matrix:
DG = RT ln ([H+]cytosol/[H+]matrix) + F DY = 2.3 RT (pHmatrix - pHcytosol) + F DY

In the absence of ADP, H+ cannot flow back to the matrix through Fo. The pH and electrical gradients (DpH & DY) are maximal. As respiration with outward H+ pumping proceeds, the free energy change for H+ ejection (positive DG) increases and approaches the magnitude of that for electron transfer (negative DG). When the coupled reaction becomes 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. 

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

Uncoupling reagents (uncouplers) are lipid-soluble weak acids.  For example, H+ (shown in red) can dissociate from the hydroxyl group of the uncoupler dinitrophenol. Diagram p. 834.

Uncouplers dissolve in the membrane, and function as carriers for H+

Uncouplers block oxidative phosphorylation by dissipating the H+ electrochemical gradient.

Protons pumped out are carried by the uncoupler back into the mitochondrial matrix, preventing development of a pH or electrical gradient.
With an uncoupler present there is no pH or electrical gradient. DG for H+ ejection is zero, and 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.

Since DG for H+ flux is zero in the absence of a H+ gradient, and hydrolysis of ATP is spontaneous, the ATP Synthase reaction runs backward in the presence of an uncoupler.

An uncoupling protein (also called thermogenin) is produced in brown adipose tissue of newborn mammals and hibernating mammals (see p. 834-835). 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. The free energy change associated with respiration is dissipated as heat. This "non-shivering thermogenesis" is costly in terms of respiratory energy unavailable for ATP synthesis, but it provides valuable warming of the organism.

Copyright 1998-2007 by Joyce J. Diwan. All rights reserved.

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