Catalytic activity of NADH-ubiquinone oxidoreductase (Complex I) in intactmitochondria - Evidence for the slow active/inactive transition

The mammalian purified dispersed NADH-ubiquinone oxidoreductase (Complex I) and the enzyme in inside-out submitochondrial particles are known to be th e slowly equilibrating mixture of the active and de-activated forms (Vinogr adov, A. D. (1998) Biochim. Biophys. Acta 1364, 169-185). We report here th e phenomenon of slow active/de-active transition in intact mitochondria whe re the enzyme is located within its natural environment being exposed to nu merous mitochondrial matrix proteins. A simple procedure for permeabilizati on of intact mitochondria by channel-forming antibiotic alamethicin was wor ked out for the "in situ" assay of Complex I activity. Alamethicin-treated mitochondria catalyzed the rotenone-sensitive NADH-quinone reductase reacti on with exogenousely added NADH and quinone-acceptor at the rates expected if the enzyme active sites would be freely accessible for the substrates. T he matrix proteins were retained in alamethicin-treated mitochondria as jud ged by their high rotenone-sensitive malate-cytochrome c reductase activity in the presence of added NAD(+). The sensitivity of Complex I to N-ethylma leimide and to the presence of Mg2+ was used as the diagnostic tools to det ect the presence of the de-activated enzyme. The NADH-quinone reductase act ivity of alamethicin-treated mitochondria was sensitive to neither N-ethylm aleimide nor Mg2+. After exposure to elevated temperature (37 degreesC, the conditions known to induce de-activation of Complex 1) the enzyme activity became sensitive to the sulfhydryl reagent and/or Mg2+. The sensitivity to both inhibitors disappeared after brief exposure of the thermally de-activ ated mitochondria with malate/glutamate, NAD(+), and cytochrome c (the cond itions known for the turnover-induced reactivation of the enzyme). We concl ude that the slow active/de-active Complex I transition is a characteristic feature of the enzyme in intact mitochondria and discuss its possible phys iological significance.