Oxidative phosphorylation: a role of lipoic acid and unsturated fatty acids.

studies with dibutyl~hloro[~H]methyltin chloride have shown that it co-migrates with subunit 9, both in its monomeric form (mol.wt. 7000-8000) and its aggregated (hexameric) form (mol.wt. 45000). Studies with yeast mitochondria and the purified ATP synthase indicate that the ATP synthase contains approx. six binding sites for triethyltin and an equivalent number for dibutylchloromethyltin chloride (K. Cain & D. E. Griffiths, unpublished work), but this accounts for less than 30 % of the dibutylchloromethyltin chloride-interaction sites in the inner membrane. Up to 35 % of the dibutylchloromethyltin chloride-interaction sites remain in the membrane when ATP synthase is extracted, and of the extractable interaction sites, 30% are not precipitated by an antibody to ATP synthase, and 5060% of the dibutylchloromethyltin chloride-interaction sites present in theTritonextract are separable from ATP synthase by density-gradient centrifugation. This suggests that the majority of the dibutylchloromethyltin chloride-interaction sites are only loosely associated with the ATP synthase complex and may form a large pool of interaction sites (20 times the concentration of F,-ATPase) which are in dynamic equilibrium with ATP synthase. Analysis of the mode of action of dibutylchloromethyltin chloride indicates that it is a specific inhibitor of oxidative phosphorylation that titrates a mobile component of the oxidative phosphorylation complex, which is present in high concentration (&lOnmol/mg of protein). The demonstration that dibutylchloromethyltin chloride reacts with a lipoic acid moiety in the mitochondrial inner membrane, the presence of lipoic acid in purified ATP synthase preparations, and the specific reversal of dibutylchloromethyltin chloride inhibition of ATPase, oxidative phosphorylation and ATP-driven energy-linked reactions by dihydrolipoic acid indicate a specific function for lipoic acid (or a lipoic acid derivative) in mitochondrial oxidative phosphorylation. Further evidence for a functional role of lipoic acid comes from the use of a Iipoic acid analogue, 8-methyl-lipoic acid, a known growth inhibitor and competitive inhibitor of lipoic acid-requiring reactions (Schmidt et al., 1969). This compound is inactive in the oxidized form, but on reduction is a good inhibitor of mitochondrial ATPase, mitochondrial oxidative phosphorylation and ATPdriven energy-linked reductions. The experiments with dibutylchloromethyltin chloride and 8-methyl-lipoic acid demonstrate clearly that lipoic acid is involved in an intermediate step in mitochondrial ATPase and mitochondrial oxidative phosphorylation involving enzymic transformation of lipoic acid.