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Independent inhibitions of mitochondrial complex V by the adenosinetriphosphatase inhibitor protein and active-site modifiers.

The methyl 4-azidobenzimidate derivative of the naturally occurring ATPase inhibitor protein (IF1) of mitochondria binds to the beta subunits of soluble F1-ATPase upon photoactivation [Klein, G., Satre, M., Dianoux, A.-C., & Vignais, P. V. (1981) Biochemistry 20, 1339--1344]. A number of specific ATPase inhibitors, namely, 4-chloro-7-nitrobenzofurazan (NBF-Cl), efrapeptin, 5'-[p-(fluorosulfonyl)benzoyl]adenosine (FSBA), phenylglyoxal, aurovertin, tridentate ferrous bathophenanthroline, and octylguanidine (referred to hereafter as "artificial" inhibitors), are also considered to bind to the beta subunit, and there is strong evidence that the first three bind at the active site. Since the inhibition by IF1 of complex V ATPase activity can be reversed by incubation of the inhibited complex at pH 8.0, this system was used to investigate whether the inhibitions brought about by IF1 and the artificial inhibitors were independent, mutually interfering, or mutually exclusive. The experiments were carried out in two ways. (a) Complex V was first maximally inhibited by IF1. Then an artificial inhibitor was added and allowed to react. Excess artificial inhibitor was removed by precipitation of the doubly inhibited complex V with ammonium sulfate and resuspension in inhibitor-free buffer at pH 8.0. Incubation at pH 8.0 released the inhibition due to IF1. However, it was found that the factor that controlled reemergence of ATPase activity was the degree of inhibition exerted by the artificial inhibitor. When the artificial inhibitor was removed first (which was done by addition of dithiothreitol when the artificial inhibitor was NBF-Cl), then reemergence of activity depended on incubation at pH 8.0 to reverse the inhibition due to IF1. These results indicated that IF1-inhibited complex V could be independently inhibited by various artificial inhibitors. The artificial inhibitors used in this type of study were NBF-Cl, efrapeptin, aurovertin, FSBA, and phenylglyoxal. (b) Complex V was first treated with the artificial inhibitor (ferrous bathophenanthroline or octylguanidine) and then with IF1. Results showed that prior treatment of complex V with these inhibitors did not interfere with IF1 subsequently exerting maximal and reversible inhibition. The above results have been discussed in view of the recent finding that F1-ATPase contains two functional and interacting hydrolytic sites [Grubmeyer, C., & Penefsky, H.S. (1981) J. Biol. Chem. 256, 3718--3727].