(H)over-right-arrow(+)/2(e)over-bar stoichiometry of the NADH : ubiquinonereductase reaction catalyzed by submitochondrial particles

Nitochondrial NADH:ubiquinone-reductase (Complex I) catalyzes proton transl ocation into inside-out submitochondrial particles. Here we describe a meth od for determining the stoichiometric ratio (H) over right arrow (+)/2e(-) (n) for the coupled reaction of NADH oxidation by the quinone accepters. Co mparison of the initial rates of NADH oxidation and alkalinization of the s urrounding medium after addition of small amounts of NADH to coupled partic les in the presence of Q(1) gives the value of n = 4. Thermally induced dea ctivation of Complex I [1, 2] results in complete inhibition of the NADH ox idase reaction but only partial inhibition of the NADH:Q(1)-reductase react ion. N-Ethylmaleimide (NEM) prevents reactivation and thus completely block s the thermally deactivated enzyme. The residual NADH:Q(1)-reductase activi ty of the deactivated, NEM-treated enzyme is shown to be coupled with the t ransmembraneous proton translocation (n = 4). Thus, thermally induced deact ivation of Complex 1 as well as specific inhibitors of the endogenous ubiqu inone reduction (rotenone, piericidin A) do not inhibit the proton transloc ating activity of the enzyme.