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

FEBS Lett. 1999 Jul 16;455(1-2):36-40. doi: 10.1016/s0014-5793(99)00850-9.

Abstract

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 IA.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 pKa 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).

Publication types

  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Animals
  • Cattle
  • Electrophoresis, Polyacrylamide Gel
  • Mitochondria, Heart / enzymology*
  • Molecular Probes
  • NAD(P)H Dehydrogenase (Quinone) / metabolism*
  • Spectrometry, Fluorescence
  • Submitochondrial Particles / enzymology
  • Sulfhydryl Compounds / metabolism*

Substances

  • Molecular Probes
  • Sulfhydryl Compounds
  • NAD(P)H Dehydrogenase (Quinone)