Properties of Higher Plant Mitochondria. III. Effects of Respiratory Inhibitors.

The effects of representative respiratory inhibitors were investigated on the coupled respiration of mung bean mitochondria using succinate and l-malate as substrates. The inhibitors studied were: (I) malonate, (II) amytal and rotenone, (III) antimycin A and 2-n-nonyl-4-hydroxyquinoline N-oxide (NOQNO), and (IV) cyanide and azide.Malonate inhibition of succinate oxidation follows a classical type of competitive inhibition with an inhibitor dissociation constant of 0.13 mm. There is no inhibition detectable when malate is used as substrate. In contrast to animal mitochondria, amytal is capable of inhibiting 20 to 40% of succinate oxidation and 90 to 100% of malate oxidation, but inhibition due to rotenone amounts to only 0 to 20% of succinate oxidation and 40 to 50% of malate oxidation. The half-maximal inhibition caused by amytal occurs at 2 to 2.5 mm and that by rotenone at 3 mmumoles/mg protein.The maximal inhibition caused by either antimycin A or NOQNO is 70 to 80% of the state 3 respiration. Very little inhibition was observed on the state 4 respiration, and both inhibitors were capable of titrating stoichiometrically with mitochondrial protein with identical titers, 0.22 mmumoles/mg protein for half-maximal inhibition. They differ, however, in that NOQNO does uncouple oxidative phosphorylation in mung bean mitochondria, but antimycin A does not do so. Both cyanide and azide inhibit the state 3 rate 65 to 80%. Inhibition of state 4 respiration can be up to 50% by cyanide, while almost none by azide. Uncoupling action was noted with cyanide, but very little with azide.It is concluded that the second state 3 rate of succinate oxidation includes 80% succinoxidase, the remaining 20% being contributed by the NADH pathway. Malate oxidation apparently does not involve succinoxidase. Malate oxidation is completely sensitive to amytal, but only 50% inhibited by rotenone. A difference between animal and plant mitochondria appears to be in the flavoproteins associated with NADH oxidation.From the observations that antimycin A, NOQNO, cyanide, and azide do not cause complete inhibition, it is suggested that a leakage of electrons to oxygen exists before the site of inhibition of antimycin A or NOQNO.