2.4.6  Oxidative phosphorylation

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When electrons are transferred from NADH to O2, a large release of redox energy enables ATP formation in complex V of the respiratory chain (ATP synthase in Figure 2.24). Energy release associated with electron transport is conserved by H+ translocation across the membrane to form a protonmotive force (ΔµH+) which has both an electrical (ψ) and a pH component (ΔµH+ = Δψ + ΔpH). This forms the basis of the chemiosmotic theory proposed by Mitchell in 1960 and now widely accepted. See Equations 6.16a and 6.16b in Nobel 1983.

In plant mitochondria, ΔµH+ exists mainly as a Δψ of 150–200 mV, with a pH gradient (ΔpH) of 0.2–0.5 units. ATP synthesis occurs as H+ move from a compartment of high potential (outside the membrane) to one of low potential (the mitochondrial matrix) through the ATP synthase complex. Oxidation of NADH via the cytochrome pathway has three H+ translocation sites associated with this process, and is linked to synthesis of up to three ATP molecules for each molecule of NADH oxidised (i.e. three ATP formed per two electrons). By contrast, both succinate and external NADH oxidation, or NADH oxidation via the rotenone-insensitive bypass, are linked to the synthesis of only two ATP molecules per two electrons, as these events are associated with only two H+ pumping sites.

The number of H+ translocated for each pair of electrons transferred from NADH to O2 remains controversial; it will depend, in the final analysis, on the mechanism(s) by which H+ translocation is coupled to electron flow and this has still to be elucidated. Given the magnitude of free energy change during ATP synthesis and the magnitude of measured ΔµH+, at least three H+ are needed per ATP synthesised. Consequently, the H+/2e ratio for oxidation of NADH must be at least 9.


Figure 2.26 Stylised O2 electrode recording of respiring plant mitochondria illustrating respiratory control. Oxygen consumption is measured as a function of time. The isolated mitochondria are depleted of substrates and are therefore dependent on added substrate. Addition of ADP (inorganic phosphate is in the reaction medium) allows oxidative phosphorylation to proceed, dissipating some of the protonmotive force and thereby stimulating electron transport; the enhanced rate of O2 uptake is called State 3. When all added ADP is phosphorylated, electron transport slows to what is known as State 4 ('resting' state). Addition of more ADP stimulates O2 uptake further, but addition of oligomycin, which blocks the ATP synthetase, lowers O2 uptake to the State 4 rate. Addition of an uncoupler (protonophore) fully dissipates the protonmotive force and stimulates O2 uptake; no ATP synthesis occurs in the presence of the uncoupler. When the O2 concentration falls to zero, respiration ceases (Original drawing courtesy David Day)

(a) ATP synthase

ATP synthase is another multi-subunit complex (Figure 1 in Case study 2.1) with at least nine polypeptides, some present in multiple copies, four of which are synthesised in the mitochondrion. This massive complex spans the inner mitochondrial mem-brane and comprises two major parts: F0, which consists of hydrophobic subunits embedded in the inner membrane and which acts as an H+ pore or channel; and F1, which is hydrophilic and extends into the matrix on a ‘stalk’. F1 contains the active site of the ATP synthase and is a reversible ATPase. When connected to F0, F1 can either hydrolyse ATP and drive H+ translocation into the intermembrane space, or, when ΔµH+ drives H+ back into the matrix through F0, it can synthesise ATP from bound ADP and Pi. The stalk contains a protein known as the oligomycin-sensitivity-conferring protein (OSCP) because it binds the antibiotic oligomycin which then prevents H+ translocation through F0 and inhibits ATP synthesis. Therefore, adding oligomycin to mitochondria oxidising a substrate in the presence of ADP restricts O2 uptake (Figure 2.26).

The reaction mechanism of the ATP synthase remains controversial but the most favoured hypothesis is a
‘con-formational’ model. According to this model, F1 has three nucleotide-binding sites which can exist in three con-figurations: one with loosely bound nucleotides, one with tightly bound nucleotides and the third in a nucleotide-free state. H+ movement through F0 results in rotation of F1, causing a conformational change during which the site with loosely bound ADP and Pi is converted to one which binds them tightly in a hydrophobic pocket in which ATP synthesis occurs. Further H+ movement then causes another rotation of F1 and the ATP binding site is exposed and releases the nucleotide. In the meantime, the other nucleotide-binding sites are undergoing similar changes, with ADP and Pi being bound and converted to ATP. Thus H+ translocation drives the three sites through three different configurations and the main expenditure of energy is in the induction of a conformational change that releases tightly bound ATP, rather than in ATP synthesis itself.

(b) Respiratory control

Electron transport through the respiratory chain, and therefore rate of O2 uptake, is controlled by availability of ADP and Pi, a phenomenon described as ‘respiratory control’. In the absence of ADP or Pi, the proton pore of ATP synthase is blocked and a ΔµH+ builds up to a point where it restricts further H+ translocation across the inner membrane. Since electron transport is functionally linked to H+ translocation, this elevated ΔµH+ will also restrict O2 uptake. That outcome is easily seen with isolated mitochondria (Figure 2.26) where O2 uptake is stimulated by adding ADP (‘State 3’ respiration). When all of the added ADP has been consumed, O2 uptake decreases again (‘State 4’). In steady state, the rate of electron flow is determined by the rate of flow of H+ back across the membrane: when ADP and Pi are available the backflow is rapid and occurs via ATP synthase; in the absence of these compounds, backflow is no more than a slow leak. The ratio of State 3 to State 4 (the respiratory control ratio) is thus an indication of coupling between ADP phosphorylation and electron transport. Larger values represent tighter coupling. The proton leak can be dramatically stimulated by some compounds which act as protonophores or proton channels; these compounds collapse the ΔµH+ and increase O2 uptake up to the State 3 rate (Figure 2.26). However, no ATP is formed and these compounds are called uncouplers because they uncouple the linked processes of electron transport and phosphorylation.