Studies on the Respiratory Chain-linked Reduced Nicotinamide Adenine Dinucleotide Dehydrogenase XIII. BINDING SITES OF ROTENONE, PIERICIDIN A, AND AMYTAL IN THE RESPIRATORY CHAIN*

The binding sites of rotenone, piericidin A, and Amytal in the reduced nicotinamide adenine dinucleotide oxidase chain of heart have been studied with the aid of rotenone6aJ4C. Binding of rotenone continues in a linear manner beyond the point of maximal inhibition of respiration, indicating that rotenone is tightly bound not only at the specific site in the NADH dehydrogenase segment of the respiratory chain but also at other sites in submitochondrial particles. Unspecific binding is reduced by treatment of the particles with rotenone in the presence of bovine serum albumin (BSA) and is further minimized by successive washing with BSA, with only minimal removal of rotenone from the specific site. RotenoneJ4C is not removed from the specific site by denaturation or proteolysis but is largely removed by repeated extractions with anhydrous acetone, which results in recovery primarily of rotenone with only small amounts of a less inhibitory oxidation product. Substantial reversal of the inhibition of respiration occurs on repeated washing of the inhibited particles with BSA. Thus, contrary to expectations, under usual assay conditions rotenone is not a specific, stoichiometric, and irreversible inhibitor. Studies involving preincubation of the electron transport particle with various unlabeled inhibitors, of widely varying chemical type and inhibitory potency, prior to addition of rotenoneJ4C for determination of its subsequent binding, provide strong