Catalytic hydrolysis and synthesis of adenosine 5'-triphosphate by stereoisomers of covalently labeled F1-adenosine triphosphatase and reconstituted submitochondrial particles

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Abstract
Bovine heart F1-adenosinetriphosphatase (F1) was labeled specifically and precisely with 7-chloro-4-nitro-2,1,3-[14C]benzoxadiazole ([14C]NBD-Cl). The stereospecfically labeled F1 (O-.beta.''-[14C]-NBD-F1) was partially reactivated by LiCl treatment, which could cause rearrangement of the .beta. subunits to form O-.beta.'',.beta."-[14C]NBD-F1. Both labeled enzymes were used to combine with F1-deficient submitochondrial particles (ASU) to form the reconstituted particles O-.beta.''-NBD-F1-ASU and O-.beta.'',.beta."-NDB-F1-ASU, respectively. A comparison of the observed steady-state rates of catalytic ATP hydrolysis and oxidative phosphorylation by these specifically labeled submitochondrial particles (SMP) with those of the unlabeled control samples suggests that oxidative phosphorylation involves more active sites of F1 than catalytic ATP hydrolysis. A comparison of the observed ATPase activity of uncoupled labeled SMP and the activity for ATP-driven reverse electron transport in coupled labeled SMP with the corresponding values of the unlabeled control samples shown that the observed fractional inhibition ATP hydrolysis is the same for both the coupled SMP and uncoupled SMP and is determined only by the state of stereospecific labeling of F1. The effect of preincubation under simulated oxidative phosphorylation conditions on the ATPase activity of the unperturbed, specifically NBD-labeled submitochondrial partricles was also examined. The data show that respiration-generated proton flux does not cause the .beta. subunits in bovine heart proton-ATPase to continue switching places with each other during oxidative phosphorylation. Samples of NBD-F1 with specific labels on its nonhydrolytic .beta." subunits but none on its hydrolytic .beta.'' subunit were prepared by a three-cycle process. Both this specifically labeled enzyme, O-.beta."-NBD-F, and the unlabeled control, F1, were used to reconstitute the submitochondrial particles. It was found that for the first time stereospecifically labeled submitochondrial particles have been prepared that exhibit a lower catalytic efficiency relative to the unlabeled control particles for ATP synthesis than ATP hydrolysis. These results led to the following conclusions: (1) Due to interaction with the smaller subunits in MF1, the three .beta. subunits become functionally distinguishable. Efficient hydrolysis of ATP takes place at the directly active site of .beta.'' subunit when and only when proper ligands are bound at the indirectly active sites of .beta." subunits. (2) Because of conformation changes driven by the proton flux, all three active sites of F0F1 catalyze ATP synthesis, though probably not with the same efficiency. (3) Internal rotation of .alpha.3.beta.3 relative to .gamma..delta..epsilon. did not occur during the steady-state oxidative phosphorylation in submitochondrial particles.