Based on published evidence that cation transport in mitochondria is not significantly dependent on a membrane potential, it is suggested that the process of mitochondrial cation transport may be nonelectrogenic. These experiments focused on the possibility that K+ flux into rat liver mitochondria may be directly coupled, via an energy-linked carrier mechanism, to OH- influx or H+ efflux. The dependence of the unidirectional K+ influx on the external K+ concentration indicates involvement of a saturable mechanism. Increasing the external pH from 7.0 to 8.0 increases the apparent V max of the K+ influx without significantly altering the apparent Km for K+. The pH dependence is greater in the presence of N-ethyl maleimide, a known inhibitor of the mitochondrial Pi/OH- exchange mechanism. N-Ethyl maleimide decreases the apparent V max at pH 7.0 and increases it at pH 8.0. Evidence indicates that both N-ethyl maleimide and a high external Pi concentration may stimulate the K+ influx at alkaline external pH (8.0) by preventing net exchanges between endogenous Pi and external OH-. An apparent first-order dependence of the K+ influx on the external OH- concentration is observed in the presence of N-ethyl maleimide. These results are consistent with a possible role of external OH- as a cosubstrate of the K+ transport mechanism.

566th MEETING, CAMBRIDGE 203 extramatrix concentrations of ATP, ADP and Pi and the matrix volume being determined in parallel. As is shown in Fig. I , the thermodynamic relation is consistent with a mean stoicheiometry of 2.66+0.01-+ H+/ATP obtained by linear regression forced through the origin. As any systematic error in matrix volume or in activity coefficients for the isotopic indicators of membrane potential or pH gradient would affect the apparent magnitude of A&+ equally for each point, it is significant that the slope of Fig. 1 not forced through the origin) is 2.61 k0.02 H+/ATP. Thus the apparent stoicheiometry remains constant as A,&,+ is decreased, and it was not possible to confirm the increase in stoicheiometry observed, under these circumstances, by Weichmann et al. ( 1 975).In conclusion, two independent lines of evidence suggest that not less than 5.4 protons cycle across the membrane to cause the synthesis of 2mol of ATP during the transfer of two electrons from a flavoprotein-linked substrate to oxygen. This paper will summarize the evidence that lipoic acid plays a key role in mitochondrial oxidative phosphorylation. In contrast with substrate-level phosphorylation, lipoic acid is the active functional group in the ATP synthase complex and is a mobile component of the mitochondria1 inner membrane, which functions as an energy-coupling factor that can link the electron-transfer chain and the ATP synthase compIex. The demonstration of this function of lipoic acid thus fulfils the basic requirements of any chemical hypothesis of oxidative phosphorylation. Materials and methodrDibutylchloromethyltin chloride and dibutylchlor~[~H]rnethyltin chloride were synthesized from dibutyltin chloride and diazomethane or dia~o[~H]methane respectively (Seyferth & Rochow 1955; K. Cain & D. E. Griffiths, unpublished work).Binding of dibutylchlor~[~HJmethyltin chloride to mitochondria, submitochondrial particles and chloroplasts was measured by addition of an excess of dib~tylchloro[~H]methyltin chloride (1&20nmol/mg of protein) to membrane preparations and washing four times with suspending medium after high-speed centrifugation. Free lipoic acid was assayed by bioassay of sterile protein fractions or acid extracts, by using a lipoic acid-VOl. 5