NEW INHIBITORS OF THE UBIQUINOL OXIDASE OF HIGHER-PLANT MITOCHONDRIA

A screen has been performed of possible inhibitors of the ubiquinol ox idase of higher plant mitochondria by assaying their effects on cyanid e-insensitive NADH oxidase of mitochondria of Arum maculatum. A number of compounds which have powerful inhibitory effects have been identif ied. Potent inhibition was found with compounds related to the previou sly described il-propyl gallate, but with the n-propyl sidechain repla ced with alkyl chains of greater hydrophobicity. Titration of a range of partial reactions showed that the inhibitors act specifically on th e ubiquinol oxidase. The concentrations of inhibitor required are depe ndent on the respiratory substrate and on the amount of mitochondria u sed in the assay. Octyl gallate also proved to be a potent inhibitor o f the ubiquinol oxidase in tobacco cell suspensions. A second class of compounds which strongly inhibit cyanide-insensitive NADH oxidation i s aurachin C and its analogues. Compounds related to aurachin D are mu ch less effective. Titrations of a range of partial reactions indicate that inhibition is caused by a direct action on the ubiquinol oxidase , However, both types of aurachins also act strongly at the Q(i) site of the cytochrome bc(1) complex, as already known to be the case in ot her systems, and so they are of more limited value for studies of the ubiquinol oxidase. Titration of the oxidation of NADH via the ubiquino l oxidase in a purified mitochondrial fraction from the spadices of Ar um maculatum with octyl gallate gave a half-maximal effect at a concen tration of around 6 nM when the protein concentration was 14 mu g ml(- 1). A similar titre was obtained with a decyl derivative of aurachin C . This allowed us to estimate an upper limit for the concentration of ubiquinol oxidase in these mitochondria of 0.72 +/- 0.15 nmol mg(-1) p rotein, or a ratio of ubiquinol oxidase/cytochrome oxidase of about 15 +/- 7:1. The measurements also provide a minimal turnover number for the ubiquinol oxidase of 186 +/- 42 electrons . s(-1). Titration of th e ubiquinol oxidase in soybean cotyledon mitochondria with these compo unds gave the concentration of inhibitor required to elicit 50% of the maximum observed effect (I-50) values about one order of magnitude hi gher than those found with Arum mitochondria, and again the values dep ended on the respiratory substrate. An explanation for the variation i n I-50 values may be found in terms of differences in oxidase concentr ations in the different mitochondrial membranes and in the differences in rate-controlling steps with substrates of different activities.