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

Abstract

Inside‐out submitochondrial particles from both potato (Solanum tuberosum L. cv. Bintje) tubers and pea (Pisum sativum L. cv. Oregon) leaves possess three distinct dehydrogenase activities: Complex I catalyzes the rotenone‐sensitive oxidation of deamino‐NADH, NDin(NADPH) catalyzes the rotenone‐insensitive and Ca2+‐dependent oxidation of NADPH and NDin(NADH) catalyzes the rotenone‐insensitive and Ca2+‐independent oxidation of NADH. Diphenylene iodonium (DPI) inhibits complex I, NDin(NADPH) and NDin (NADH) activity with a Ki of 3.7, 0.17 and 63 µM, respectively, and the 400‐fold difference in Ki between the two NDin made possible the use of DPI inhibition to estimate 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 µM DPI. The maximum decrease in rate was 10–20 nmol O2 mg−1 min−1. The reduction level of NAD(P) was manipulated by measuring malate oxidation in state 3 at pH 7.2 and 6.8 and in the presence and absence of an oxaloacetate‐removing system. The inhibition by DPI was largest under conditions of high NAD(P) reduction. Control experiments showed that 125 µM DPI had no effect on the activities of malate dehydrogenase (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 µM 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 mitochondria oxidizing malate is due to inhibition of NDin(NADPH) oxidizing NADPH. Thus, NADP turnover contributes to malate oxidation by plant mitochondria.