Interactions of gamma T273 and gamma E275 with the beta subunit PSAV segment that links the gamma subunit to the catalytic site Walker homology B aspartate are important to the function of Escherichia coli F1F0 ATP synthase.
In Escherichia coli F(1)F(o) ATP synthase, gammaT273 mutants that eliminate the ability to form a hydrogen bond to betaV265 were incapable of ATP synthase-dependent growth and ATPase-dependent proton pumping, had very low rates of ATPase activity catalyzed by purified F(1), and had significantly decreased sensitivity to inhibition by Mg(2+)-ADP-AlF(n) species, while gammaT273D and gammaT273N mutants which maintained or increased the hydrogen bond strength maintained or increased catalytic activity. The betaP262G mutation that increases the potential flexibility of the rigid sleeve that surrounds the gamma subunit C-terminus also virtually eliminated ATPase activity and susceptibility to Mg(2+)-ADP-AlF(n) inhibition. The gammaE275 mutants that retained the ability to form the betaV265 hydrogen bond had higher ATPase activity than those that eliminated the hydrogen bond. These results provide evidence that the ability to form hydrogen bonds between betaV265 and the gamma subunit C-terminus contributes significantly to the rate-limiting step of catalysis and to the ability of the F(1)F(o) ATP synthase to use a proton gradient to drive ATP synthesis. The loss of activity observed with betaP262G may result from increased flexibility conferred by glycine that decreases the efficiency of communication between the gamma subunit-betaV265 hydrogen bonds and the Walker B aspartate at the catalytic site. The partial loss of coupling observed with gammaT273 mutants that eliminate the betaV265 hydrogen bond is consistent with participation of this hydrogen bond in the escapement mechanism for ATP synthesis in which interactions between the gamma subunit and (alphabeta)(3) ring prevent rotation until the empty catalytic site binds substrate.
Center for the Study of Early Events in Photosynthesis, Faculty of Biomedicine and Biotechnology, School of Life Sciences, Arizona State University, Tempe, Arizona 85287-4501, USA.
Kathryn W
KW
Boltz
Wayne D
WD
Frasch
D000255:Adenosine Triphosphate / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism; D000818:Animals; D001224:Aspartic Acid / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D025243:Bacterial Proton-Translocating ATPases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D002384:Catalysis; D002417:Cattle; D025222:Chloroplast Proton-Translocating ATPases / Q000737:chemistry / Q000378:metabolism; D017124:Conserved Sequence; D029968:Escherichia coli Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D006860:Hydrogen Bonding; D006868:Hydrolysis; D025261:Mitochondrial Proton-Translocating ATPases / Q000737:chemistry / Q000378:metabolism; D016297:Mutagenesis, Site-Directed; D010446:Peptide Fragments / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D021122:Protein Subunits / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D018892:Proton-Motive Force / Q000235:genetics; D018724:Spinacia oleracea; D040681:Structural Homology, Protein; D013379:Substrate Specificity / Q000235:genetics; D013816:Thermodynamics
D029968:Escherichia coli Proteins; D010446:Peptide Fragments; D021122:Protein Subunits; D001224:Aspartic Acid; D000255:Adenosine Triphosphate; D025243:Bacterial Proton-Translocating ATPases; D025222:Chloroplast Proton-Translocating ATPases; D025261:Mitochondrial Proton-Translocating ATPases