Topography and chemical reactivity of the active-inactive transition-sensitive SH-group in the mitochondrial NADH:ubiquinone oxidoreductase (Complex I).
The spatial arrangement and chemical reactivity of the activation-dependent thiol in the mitochondrial Complex I was studied using the membrane penetrating N-ethylmaleimide (NEM) and non-penetrating anionic 5,5'-dithiobis-(2-nitrobenzoate) (DTNB) as the specific inhibitors of the enzyme in mitochondria and inside-out submitochondrial particles (SMP). Both NEM and DTNB rapidly inhibited the de-activated Complex I in SMP. In mitochondria NEM caused rapid inhibition of Complex I, whereas the enzyme activity was insensitive to DTNB. In the presence of the channel-forming antibiotic alamethicin, mitochondrial Complex I became sensitive to DTNB. Neither active nor de-activated Complex I in SMP was inhibited by oxidized glutathione (10 mM, pH 8.0, 75 min). The data suggest that the active/de-active transition sulfhydryl group of Complex I which is sensitive to inhibition by NEM is located at the inner membrane-matrix interface. These data include the sidedness dependency of inhibition, effect of pH, ionic strength, and membrane bilayer modification on enzyme reactivity towards DTNB and its neutral analogue.
Gostimskaya
Irina S
IS
Department of Biochemistry, School of Biology, Moscow State University, Moscow 119992, Russian Federation.
Cecchini
Gary
G
Vinogradov
Andrei D
AD
D000818:Animals; D002417:Cattle; D004228:Dithionitrobenzoic Acid / Q000378:metabolism; D042967:Electron Transport Complex I / Q000737:chemistry* / Q000378:metabolism*; D005033:Ethylmaleimide / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008929:Mitochondria, Heart / Q000201:enzymology*; D010539:Permeability; D051381:Rats; D013329:Structure-Activity Relationship; D013367:Submitochondrial Particles / Q000378:metabolism; D013438:Sulfhydryl Compounds / Q000737:chemistry* / Q000378:metabolism*
D013438:Sulfhydryl Compounds; D004228:Dithionitrobenzoic Acid; D042967:Electron Transport Complex I; D005033:Ethylmaleimide