5.7.1.4—
Mechanism of Coupling

The mechanism of energy coupling remains an intractable problem in spite of the impressive array of workers in this area. Three outstanding hypotheses are under active consideration: (a) the chemical intermediate hypothesis; (b) the chemiosmotic hypothesis; and (c) the conformation coupling hypothesis.

The operation of the chemical intermediate hypothesis may be summarized as follows:

where A and B are adjacent electron carries at the coupling site, X and I are unknown couplers common to all coupling sites (see Greville, 1969). The scheme postulates the existence of both non-phosphorylated and phosphorylated intermediates. A non-phosphorylated intermediate accounts for the action of uncouplers, which is independent of the presence of phosphate. The nonphosphorylated intermediate can also be coupled to work functions such as ion transport, which is sensitive to uncouplers, but not to the phosphorylation inhibitor, oligomycin. The phosphorylated intermediate transfers a phosphoryl group to ADP. Hill and Boyer (1967) showed that the bridge oxygen between the b and g phosphorus of ATP is furnished by ADP. Hence, the mechanism of phosphorylation involves an activation of phosphate.

The chemiosmotic hypothesis in its simplest form as first proposed by Mitchell (1961) involves a vectorial metabolism in which the elements of water are transported to opposite sides of the mitochondrial membrane (Fig. 5.5).

Figure 5.5
Representation of the Mitchell chemiosmotic
scheme for oxidative phosphorylation.
(Redrawn from Mitchell, 1966.)

Thus oxido-reduction reactions create a pH gradient as well as a potential difference (outside positive) which tends to drive H⁺ back across the membrane into the inner compartment. This force is the proton-motive force, and it is this flow of protons through the coupling site (a reversible ATPase) which drives ATP synthesis. The phosphorylation reaction is represented as follows:

It can then be shown that in the absence of a transmembrane electrical potential, a pH differential of 3.5 units is required to poise the ratio of ATP/ADP = 1 while a potential difference of 210 mV would be required in the absence of pH gradients (Mitchell, 1966). The translocation of protons and equivalent OH– is effected by ionizable groups which are designated XH and IOH, corresponding to components of an ATPase. The proposed intermediate X-I of the ATPase must have a sufficiently low hydrolysis constant at the high electrochemical

potential of H⁺ in the outer phase to come to reverse equilibrium with water according to the reaction

On the other hand, the hydrolysis constant of the intermediate X ~ I must be in the order of 10⁵M , and the intermediate X ~ I must be in equilibrium with the ATP/(ADP + P_i ) couple in the inner phase, so that

The system vibrates between states in which the intermediate X – I is alternately accessible to the outer and inner phases, being X – I when in contact with the outer phase and X – I when in contact with the inner phase. The transition of X – I to X ~ I is due not to the pumping of energy into X–I, but to a lowering of the ground state energy for X – I hydrolysis by some 10,000 calories per mole on translocation through an anisotropic membrane.

While experimental verification of the chemiosmotic mechanism has been realized only in chloroplasts by acid-base trasitions (Jagendorf & Uribe, 1966; see also chapter 4), similar experiments have not been successful in mitochondria. Cockrell et al., (1967) have shown ATP synthesis after establishing a K+ gradient by valinomycin-treated mitochondria. However, Glynn (1967) argued that ATP synthesis via a K⁺ gradient could be explained equally well on the basis of a membrane potential rather than cation transport down a chemical gradient through an ATPase.

In their general aspects, the chemical intermediate hypothesis and the chemiosmotic hypothesis differ primarily in the nature of the initial driving force for coupled phosphorylation. The two hypotheses converge at the level of the ATPase in that both call for an unknown intermediate, X ~ I (Greville, 1969).

The conformational coupling hypothesis of Boyer (1967) differs in that the ATPase has a high affinity for P_i and ADP. Tightly bound ATP is formed via a nucleophilic attack by ADP on orthophosphate in a S_N² reaction with the displacement of water (Korman & McLick, 1970). According to the model, there exists an energy requirement for the release of bound ATP from the complex, by changing the complex from one having a high affinity for ATP to one having low affinity (Boyer et al., 1973). The advantages of this hypothesis are that it explains most of the exchange reactions observed in coupled phosphorylation, and the coupling of ATP to energy yielding reactions of mitochondria.