Specific labelling of a constituent polypeptide of bovine heart mitochondrial reduced nicotinamide-adenine dinucleotide-ubiquinone reductase by the inhibitor diphenyleneiodonium

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Abstract
NADH-ubiquinone-1 and NADH-menadione reductase activities of Complex I were inhibited by diphenyleneiodonium (apparent Ki 23 and 30 nmol/mg of protein respectively). Reduction of K3Fe(CN)6 and juglone was relatively unaffected. Iodoniumdiphenyl and derivatives were much less effective inhibitors. Compounds with similar ring structures to diphenyleneiodonium, in particular dibenzofuran, were inhibitors of NADH-ubiquinone-1 oxidoreductase. Diphenylene[125I]iodonium specifically labeled a polypeptide of MW 23,500. Maximum incorporation was 1 mol/mol of Complex-I flavin or 1 mol/mol of the MW 23,500 polypeptide. The label associated with this polypeptide was of limited stability, especially at lower pH. Complete inhibition of ubiquinone reduction was achieved when 1 mol of inhibitor was incorporated/mol of Complex-I flavin, but the relationship between inhibition and labeling was not linear. No evidence for covalent interaction between diphenyleneiodonium and the phospholipids of Complex I was obtained. Rotenone increased the apparent affinity of diphenyleneiodonium for the MW 23,500 polypeptide without affecting the maximum incorporation. The MW 23,500 polypeptide was not solubilized by chaotropic agents. Prior treatment of Complex I with chaotropic agents or sodium dodecyl sulfate prevented incorporation of diphenyleneiodonium into this polypeptide.