The proximal N-terminal amino acid residues are required for the coupling activity of the bovine heart mitochondrial factor B

Treatment of the recombinant bovine factor B with trypsin yielded a fragment (amino acid residues 62-175) devoid of coupling activity . Removal of the N-terminal Trp2-Gly3-Trp4 peptide resulted in a significant loss of coupling activity in the FB Delta w2- w(4) deletion mutant. Sucrose density gradient centrifugation demonstrated co-sedimentation of recombinant factor B with the ADP/ATP carrier, which is present in preparations of H+-translocating F0F1-ATPase, but not in preparations of complex V. The N-terminally truncated factor B mutant FB Delta w (2)-w(4) did not co-sediment with the ADP/ATP carrier. Recombinant factor B co-sedimented with partially purified membrane sector F-0, extracted from F-1-stripped bovine submitochondrial particles with n-dodecyl-beta-D-maltoside. Factor B inhibited the passive proton conductance catalyzed by F0 reconstituted into asolectin liposomes. A factor B mutant, bearing a photoreactive unnatural amino acid pbenzoyl-L-phenylalanine (pBpa) substituted for Trp2, cross-linked with F-0 subunits e and g as well as the ADP/ATP carrier. These results suggest that the N-terminal domain and, in particular, the proximal N-terminal amino acids are important for the coupling activity and protein-protein interactions of bovine factor B. (c) 2008 Elsevier Inc. All rights reserved.