On the reaction of piericidin A with the reduced nicotinamide adenine dinucleotide dehydrogenase of Candida utilis

The characteristics of the inhibition of the respiratory chain linked NADH dehydrogenase (nicotinamide adenine dinucleotide dehydrogenase) of C. utilis by piericidin A, and the binding of this inhibitor, were studied in inner membrane preparations with the aid of [¹⁴C]piericidin A. As in beef heart preparations, the binding of piericidin by the particles was linear and extended considerably beyond the range of maximal inhibition of NADH oxidation, suggesting the existence of unspecific binding sites. Washing of the particles with bovine serum albumin, following inhibition with piericidin, or titrating with piericidin in the presence of bovine serum albumin, did not distinguish the specific binding sites involved in the inhibition from unspecific ones, as in the case ofbeef heart preparations, because bovine serum albumin dissociated the inhibitor from all sites and caused virtually complete release of the inhibition. The apparently lower affinity for piericidin binding of sites concerned with inhibition of NADH oxidation prevented accurate determination of their number per mole of NADH dehydrogenase. From the amount of piericidin required for maximal inhibition of NADH oxidase and from Hill plots, it was clear that more than one binding site was involved in complete inhibition, and available evidence suggested that there might be 2 sites, as in animal tissues. Spectrophotometric studies of the oxidation reduction cycle of NADH dehydrogenase in C. utilis gave results comparable to those reported for beef heart preparations. These showed, in accord with the effect of piericidin on the reaction of the enzyme with various electron acceptors, that this inhibitor blocked electron flux in Candida between iron sulfur Center 2 of the enzyme (Site 1 of energy conservation) and the coenzyme Q pool, as in animal tissues.