Solubilization of the alternative oxidase of cuckoo-pint (Arum maculatum) mitochondria. Stimulation by high concentrations of ions and effects of specific inhibitors

Abstract

Selective solubilization of cyanide- and antimycin-insensitive duroquinol oxidase activity from cuckoo-pint (A. maculatum) mitochondria was achieved using taurocholate. Inhibitor-sensitivities and water-forming DQH2 (tetramethyl-p-hydroquinone, reduced form): O2 stoichiometry were the same for the alternative oxidase of intact mitochondria. Cyanide-insensitive oxidation of DQH2 by intact and solubilized mitochondria was stimulated up to 4-fold by high concentrations of anions high in the Hofmeister series, such as phosphate, sulfate or citrate. Optimal (0.7 M) sodium citrate increased Vmax for DQH2 oxidation by the solubilized preparation from 450-2400 nmol of O2 .cntdot. min-1 .cntdot. mg of protein-1 and decreased the apparent Km for DQH2 from 0.53-0.38 mM. Inhibition of solubilized DQH2 oxidase activity by CLAM (m-chlorobenzhydroxamic acid) and SHAM (salicylhydroxamic acid) was mixed competitive/non-competitive, with apparent inhibition constants for CLAM of 25 .mu.M (Ki) and 81 .mu.M (Ki) and for SHAM of 53 .mu.M (Ki) and 490 .mu.M (Ki). Propylgallate and UHDBT were non-competitive inhibitors with respect to DQH2 (apparent Ki = 0.3 .mu.M and 12 nM, respectively). Low concentrations of C18 fatty acids selectively inhibited cyanide-insensitive oxidation by intact and solubilized mitochondria, and inhibition was reversed by 1% (wt/vol) bovine serum albumin. Inhibition was competitive with DQH2, suggesting that fatty acids interfere reversably with the binding of DQH2 to the oxidase. These results support the view that quinol oxidation by the alternative pathway of A. maculatum mitochondria is catalyzed by a quinol oxidase protein, rather than by a non-enzymic mechanism involving fatty acid peroxidative reactions.