Rotation of subunits during catalysis by Escherichia coli F1-ATPase.
During oxidative and photo-phosphorylation, F0F1-ATP synthases couple the movement of protons down an electrochemical gradient to the synthesis of ATP. One proposed mechanistic feature that has remained speculative is that this coupling process requires the rotation of subunits within F0F1. Guided by a recent, high-resolution structure for bovine F1 [Abrahams, J. P., Leslie, A. G., Lutter, R. & Walker, J. E. (1994) Nature (London) 370, 621-628], we have developed a critical test for rotation of the central gamma subunit relative to the three catalytic beta subunits in soluble F1 from Escherichia coli. In the bovine F1 structure, a specific point of contact between the gamma subunit and one of the three catalytic beta subunits includes positioning of the homolog of E. coli gamma-subunit C87 (gamma C87) close to the beta-subunit 380DELSEED386 sequence. A beta D380C mutation allowed us to induce formation of a specific disulfide bond between beta and gamma C87 in soluble E. coli F1. Formation of the crosslink inactivated beta D380C-F1, and reduction restored full activity. Using a dissociation/reassembly approach with crosslinked beta D380C-F1, we incorporated radiolabeled beta subunits into the two noncrosslinked beta-subunit positions of F1. After reduction of the initial nonradioactive beta-gamma crosslink, only exposure to conditions for catalytic turnover results in similar reactivities of unlabeled and radiolabeled beta subunits with gamma C87 upon reoxidation. The results demonstrate that gamma subunit rotates relative to the beta subunits during catalysis.
D000595:Amino Acid Sequence; D002384:Catalysis; D004220:Disulfides; D004926:Escherichia coli / Q000201:enzymology; D046911:Macromolecular Substances; D008969:Molecular Sequence Data; D009038:Motion; D011487:Protein Conformation; D006180:Proton-Translocating ATPases / Q000737:chemistry / Q000378:metabolism*; D013329:Structure-Activity Relationship
D004220:Disulfides; D046911:Macromolecular Substances; D006180:Proton-Translocating ATPases