The N-termini of the α and β subunits at the top of F1 stabilize the energy-transfer function in the mitochondrial F1F0 ATP synthase

Limited trypsin digestion of isolated F-1 removed 15 and 7 amino acids from the N-termini of the alpha and beta subunits respectively and left other subunits untouched as shown by electrophoresis, immunoblotting and protein sequencing. The cooperativity for ATP hydrolysis by soluble F-1 was impaired by trypsin digestion. The K-m2 obtained from Eadie-Hofstee plots apparently decreased in trypsin-digested F-1 but the affinity for adenosine 5'-[beta,gamma-imido]triphosphate (AdoPP[NH]P) and GTP hydrolysis was not influenced. The inhibition of ATP hydrolysis by ADP was attenuated by trypsin digestion. Trypsin digestion of F-1 did not affect its capacity to bind to F-o nor did it alter the sensitivity of ATP hydrolysis in the F1Fo reconstituted system to oligomycin and N,N'-dicyclohexylcarbodiimide. The cleavage of the alpha and beta subunits did, on the other hand impair: (a) the ATP-driven proton pumping in the reconstituted F1Fo complex; (b) the inhibition by F-1 of passive proton conduction in F-o; (c) the inhibition of passive proton conduction in F-o by AdoPP[NH]P binding to F-1. These results show that the limited cleavage of the N-termini of the alpha and beta subunits, located on the top of F-1, results in decoupling of catalysis from proton transport. The possible relationship of these observations with the binding change rotatory model of the F1Fo ATP synthase is discussed.