The internal rotenone-insensitive NADPH dehydrogenase contributes to malate oxidation by potato tuber and pea leaf mitochondria

Inside-out submitochondrial particles from both potato (Solanum tuberosum L . cV. Bintje) tubers and pea (Pisum sativum L. cv. Oregon) leaves possess t hree distinct dehydrogenase activities: Complex I catalyzes the rotenone-se nsitive oxidation of deamino-NADH. NDin(NADPH) catalyzes the rotenone-insen sitive and Ca2+-dependent oxidation of NADPH and NDin(NADH) catalyzes the r otenone-insensitive and Ca2+-independent oxidation of NADH. Diphenylene iod onium (DPI) inhibits complex I, NDin(NADPH) and NDin(NADH) activity with a K-i of 3.7, 0.17 and 63 mu M, respectively, and the 400-fold difference in K-i between the two NDin made possible the use of DPI inhibition to estimat e NDin(NADPH) contribution to malate oxidation by intact mitochondria. The oxidation of malate in the presence of rotenone by intact mitochondria from both species was inhibited by 5 mu M DPI. The maximum decrease in rate was 10-20 nmol O-2 mg(-1) min(-1). The reduction level of NAD(P) was manipulat ed by measuring malate oxidation in state 3 at pH 7.2 and 6.5 and in the pr esence and absence of an oxaloacetate-removing system. The inhibition by DP I was largest under conditions of high NAD(P) reduction. Control experiment s showed that 125 mu M DPI had no effect on the activities of malate dehydr ogenase (with NADH or NADPH) or malic enzyme (with NAD(+) or NADP(+)) in a matrix extract from either species. Malate dehydrogenase was unable to use NADP(+) in the forward reaction. DPI at 125 mu M did not have any effect on succinate oxidation by intact mitochondria of either species. We conclude that the inhibition caused by DPI in the presence of rotenone in plant mito chondria oxidizing malate is due to inhibition of NDin(NADPH) oxidizing NAD PH. Thus, NADP turnover contributes to malate oxidation by plant mitochondr ia.