Uncoupling of oxidative phosphorylation: different effects of lipophilic weak acids and electrogenic ionophores on the kinetics of ATP synthesis

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Abstract
Previous studies from this laboratory have shown that the kinetics of ATP synthesis by bovine heart submitochondrial particles (SMP) are modulated by the coupled rate of respiration between two extremes of Vmax and apparent Km''s for ADP and Pi [Matsuno-Yagi, A., and Hatefi, Y. (1986) J. Biol. Chem. 261, 14031-14038; Hekman, C., Matsuno-Yagi, A., and Hatefi, Y (1988) Biochemistry 27, 7559-7565]. Thus, with ADP as the variable substrate, ATP synthesis occurred with Vmax = 200 nmol of ATP min-1 (mg of protein)-1 at 30.degree. C and an apparent KmADP = 2-4 .mu.M at low rates of respiration, and with Vmax = 11,000 nmol of ATP min-1 (mg of protein)-1 at 30.degree. C and an apparent KmADP = 120-160 .mu.M at high rates of respiration. At intermediate respiration rates, it was necessary to introduce a third intermediate KmADP for best fit of the kinetic data, indicating that transition from one kinetic extreme to the other is not abrupt and involves intermediate kinetic states of the ATP synthase complexes. The present paper shows that uncouplers affect the kinetics of ATP synthesis by SMP in two ways. When used at moderate concentrations, electrogenic ionophores such as gramicidin D or valinomycin plus nigericin decreased the Vmax for ATP synthesis without changing the contributions of the low, intermediate, and high KmADP to the overall rate of ATP synthesis. By contrast, potent lipophilic weak acid uncouplers, such as FCCP, CCCP, S-13, and SF6847, decreased Vmax and converted the kinetics of ATP synthesis toward high KmADP. Similar results were obtained when Pi was the variable substrate, or when the energy-linked reaction studied was ATP-driven reverse electron transfer from succinate to NAD, with NAD as the variable substrate. When the ATP synthase complexes of SMP were fractionally inactivated by dicyclohexylcarbodiimide, and as a result the kinetics of ATP synthesis by these particles were converted to the high-Km mode, then partial uncoupling of oxidative phosphorylation by FCCP resulted in large increases in the apparent Km for ADP and Pi. These results have been interpreted as follows. In the absence of uncouplers, increases in the apparent KmADP and KmPi are associated with increased rates of coupled respiration and increased rates of proton flux through the ATP synthase complexes. Lipophilic weak acid uncouplers, but not gramicidin D and valinomycin plus nigericin when used at moderate uncoupling concentrations, react with the ATP synthase complexes and increase slippage in the coupling mechanism within the enzyme complex. As a result, uncoupled proton flux through the ATP synthase complex increases and results in increased apparent Km values for ADP and Pi even though the rate of ATP synthesis decreases. A similar interpretation applies to the uncoupler-induced increase in the apparent KmNAD during ATP-driven reverse electron transfer from succinate to NAD. This interpretation is also consistent with the very high apparent KmADP and KmPi obtained when SMP containing fractionally inactivated ATP synthases were partially uncoupled by FCCP. In these SMP preparations, the remaining, active ATP synthase complexes turn over very rapidly during oxidative phosphorylation [Matsuno-Yagi, A., and Hatefi, Y. (1988) Biochemistry 27, 335-340]. Partial uncoupling by a lipophilic weak acid, such as FCCP, further increases the proton flux through these active ATP synthases via the slip mechanism, thus resulting in very high apparent Km values for ADP and Pi.

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