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

FEBS Letters

Gavrikova, E.V¹  Vinogradov, A.D¹ 

[1] Department of Biochemistry, School of Biology, Moscow State University, Moscow 119899, Russia

关键词: NADH-ubiquinone oxidoreductase;    Complex I;    Respiratory chain;    Fluorescence labeling;    Bovine heart mitochondrion;    NEM;    N-ethylmaleimide;    FM;    N-fluorescein maleimide;    FP;    three-subunit flavo-iron-protein derived from complex I;    IP;    iron-sulfur-enriched protein fraction derived from complex I;    SDS;    sodium dodecyl sulfate;    SMP;    submitochondrial particles;    BSA;    bovine serum albumin;    DTNB;    dithiobis-nitrobenzoic acid;    DTT;    dithiothreitol;    Q1;    2;    3-dimethoxy-5-methyl-6-isoprenyl-1;    4-benzoquinone;   

DOI  :  10.1016/S0014-5793(99)00850-9

学科分类：生物化学/生物物理

来源: John Wiley & Sons Ltd.

【 摘 要 】

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 pK _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).

【 授权许可】

Unknown