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

• Published in: FEBS letters 1999-07, Vol.455 (1), p.36-40
• Main Authors: Gavrikova, E.V, Vinogradov, A.D
• Format: Article
• Language: English
• Subjects: Animals; Bovine heart mitochondrion; BSA, bovine serum albumin; Cattle; Complex I; DTNB, dithiobis-nitrobenzoic acid; DTT, dithiothreitol; Electrophoresis, Polyacrylamide Gel; Fluorescence labeling; FM, N-fluorescein maleimide; FP, three-subunit flavo-iron-protein derived from complex I; IP, iron-sulfur-enriched protein fraction derived from complex I; Mitochondria, Heart - enzymology; Molecular Probes; NAD(P)H Dehydrogenase (Quinone) - metabolism; NADH-ubiquinone oxidoreductase; NEM, N-ethylmaleimide; Q1, 2,3-dimethoxy-5-methyl-6-isoprenyl-1,4-benzoquinone; Respiratory chain; SDS, sodium dodecyl sulfate; SMP, submitochondrial particles; Spectrometry, Fluorescence; Submitochondrial Particles - enzymology; Sulfhydryl Compounds - metabolism
• ISSN: 0014-5793; 1873-3468
• DOI: 10.1016/S0014-5793(99)00850-9

The sensitivities of NADH oxidase and/or NADH-ubiquinone reductase activities of submitochondrial particles and purified complex I towards N-ethylmaleimide (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 was resistant to the inhibitors. The pH profile of the pseudo-first order inhibition rate suggested a p K a of about 10 for the de-activation-dependent, NEM-reactive sulfhydryl group. NADH-ubiquinone reductase of activated particles treated with an excess of NEM followed by removal of the inhibitor was still capable of slow reversible active/de-active transition. When active, NEM-treated particles were de-activated and further inhibited by N-fluorescein maleimide, specific incorporation of the fluorescence label into low molecular mass polypeptide was evident. Comparison of the specific fluorescence labeling of submitochondrial particles, crude and purified complex I showed that the active/de-active state-dependent SH-group is located in a 15 kDa polypeptide (most likely in the 15 kDa IP subunit of the iron-sulfur protein-containing fraction of complex I).