The H+ F0F1-ATP synthase complex of coupling membranes converts the proton-motive Force into rotatory mechanical energy to drive ATP synthesis. The F-1 moiety of the complex protrudes at the inner side of the membrane, the F-0 sector spans the membrane reaching the Outer side. The IF1 component of the mitochondrial complex is a basic 10 kDa protein, which inhibits the F0F1-ATP hydrolase activity. The mitochondrial matrix pH is the critical factor for the inhibitory binding of the central segment of IF1 (residue 42-58) to the F-1-alpha/beta. We have analyzed the effect of native purified IF1 the IF1-(42-58) synthetic peptide and its mutants on proton conduction, driven by ATP hydrolysis or by [K+] gradients, in bovine heart inside-out submitochondrial Particles and in liposome-reconstituted F0F1 conplex. The results show that IF1, and in particular its central 42-58 segment, displays different inhibitory affinity for proton conduction from the F-1 to the F-0 side and in the opposite direction. Cross-linking of IF1 to F-1-alpha/beta subunits inhibits the ATP-driven H+ translocation but enhances H+ conduction in the reverse direction. These observation are discussed in terms of the rotary mechanism of the F0F1 complex. (C) 2009 Elsevier Inc. All rights reserved.