Active/de-active state transition of the mitochondrial complex I as revealed by specific sulfhydryl group labeling

The sensitivities of NADH oxidase and/or NADH-ubiquinone reductase activiti es of submitochondrial particles and purified complex I towards N-ethylmale imide (NEM) and other SH-reagents were studied, Only thermally de-activated preparations [A.D. Vinogradov (1998) Biochim, Biophys, Acta 1364, 169-185] were inhibited by SH-reagents whereas the redox-pulsed, activated enzyme w as resistant to the inhibitors. The pH profile of the pseudo-first order in hibition rate suggested a pK(a) of about 10 for the de-activation-dependent , NEM-reactive sulfhydryl group, NADH-ubiquinone reductase of activated par ticles treated with an excess of NEM followed by removal of the inhibitor w as still capable of slow reversible active/de-active transition, When activ e, NEM-treated particles were de-activated and further inhibited by N-fluor escein maleimide, specific incorporation of the fluorescence label into low molecular mass polypeptide was evident, Comparison of the specific fluores cence labeling of submitochondrial particles, crude and purified complex I showed that the active/de-active state-dependent SH-group is located in a 1 5 kDa polypeptide (most likely in the 15 kDa IP subunit of the iron-sulfur protein-containing fraction of complex I), (C) 1999 Federation of European Biochemical Societies.