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(DeltaW)(2)(-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 F(0)F(1)-ATPase, but not in preparations of complex V. The N-terminally truncated factor B mutant FB(DeltaW)(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 F(0) 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.
Belogrudov
Grigory I
GI
Department of Physiology, David Geffen School of Medicine, University of California at Los Angeles, VA Greater Los Angeles Healthcare System, Rm. 324, Los Angeles, CA 90073, USA. gbelo@ucla.edu
D000595:Amino Acid Sequence / Q000235:genetics; D000818:Animals; D002417:Cattle; D003432:Cross-Linking Reagents / Q000737:chemistry; D008929:Mitochondria, Heart / Q000201:enzymology*; D025261:Mitochondrial Proton-Translocating ATPases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D010086:Oxidative Phosphorylation Coupling Factors / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D010446:Peptide Fragments / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D017384:Sequence Deletion; D013329:Structure-Activity Relationship; D013367:Submitochondrial Particles / Q000201:enzymology / Q000378:metabolism
D003432:Cross-Linking Reagents; D010086:Oxidative Phosphorylation Coupling Factors; D010446:Peptide Fragments; C032410:coupling factor B; D025261:Mitochondrial Proton-Translocating ATPases