The Respiratory Chain of Plant Mitochondria: VIII. Reduction Kinetics of the Respiratory Chain Carriers of Mung Bean Mitochondria with Reduced Nicotinamide Adenine Dinucleotide.

Addition of 90 micromolar reduced nicotinamide adenine dinucleotide (NADH) in the presence of cyanide to a suspension of aerobic mung bean (Phaseolus aureus) mitochondria depleted with ADP and uncoupler gives a cycle of reduction of electron transport carriers followed by reoxidation, as NADH is oxidized to NAD(+) through the cyanide-insensitive, alternate oxidase by excess oxygen in the reaction medium. Under these conditions, cytochrome b(553) and the nonfluorescent, high potential flavoprotein Fp(ha) of the plant respiratory chain become completely reduced with half-times of 2.5 to 2.8 seconds for both components. Reoxidation of flavoprotein Fp(ha) on exhaustion of NADH is more rapid than that of cytochrome b(553). There is a lag of 1.5 seconds after NADH addition before any reduction of ubiquinone can be observed, whereas there is no lag perceptible in the reduction of flavoprotein Fp(ha) and cytochrome b(553). The half-time for ubiquinone reduction is 4.5 seconds, and the extent of reduction is 90% or greater. About 30% of cytochrome b(557) is reduced under these conditions with a half-time of 10 seconds; both cytochrome b(562) and the fluorescent, high potential flavoprotein Fp(hf) show little, if any, reduction. The two cytochromes c in these mitochondria, c(547) and c(549), are reduced in synchrony with a half-time of 0.8 second. These two components are already 60% reduced in the presence of cyanide but absence of substrate, and they become completely reduced on addition of NADH. These results indicated that reducing equivalents enter the respiratory chain from exogenous NADH at flavoprotein Fp(ha) and are rapidly transported through cytochrome b(553) to the cytochromes c; once the latter are completely reduced, reduction of ubiquinone begins. Ubiquinone appears to act as a storage pool for reducing equivalents entering the respiratory chain on the substrate side of coupling site 2. It is suggested that flavoprotein Fp(ha) and cytochrome b(553) together may act as the branching point in the plant respiratory chain from which forward electron transport can take place to oxygen through the cytochrome chain via cytochrome oxidase, or to oxygen through the alternate, cyanide-insensitive oxidase via the fluorescent, high potential flavoprotein Fp(hf).