Oxidation of the α3(βD311C/R333C)3γ subcomplex of the thermophilic Bacillus PS3 F1-ATPase indicates that only two β subunits can exist in the closed conformation simultaneously

Abstract

In the crystal structure of the bovine heart mitochondrial F1-ATPase (Abrahams, J.P., Leslie, A. G. W., Lutter, R., and Walker, J. E. (1994) Nature 370, 621-628), the two liganded β subunits, one with MgAMP-PNP bound to the catalytic site (β(T)) and the other with MgADP bound (β(D)) have closed conformations. The empty β subunit (β(E)) has an open conformation. In β(T) and β(D), the distance between the carboxylate of β-Asp315 and the guanidinium of β-Arg337 is 3.0-4.0 Å. These side chains are at least 10 Å apart in β(E). The α3(βD311C/R333C)3γ subcomplex of TF1 with the corresponding residues substituted with cysteine has very low ATPase activity unless it is reduced prior to assay or assayed in the presence of dithiothreitol. The reduced subcomplex hydrolyzes ATP at 50% the rate of wild-type and is rapidly inactivated by oxidation by CuCI2 with or without magnesium nucleotides bound to catalytic sites. Titration of the subcomplex with iodo[14C]acetamide after prolonged treatment with CuCI2 in the presence or absence of 1 mM MgADP revealed nearly two free sulfhydryl groups/mol of enzyme. Therefore, one pair of introduced cysteines is located on a β subunit that exists in the open or partially open conformation even when catalytic sites are saturated with MgADP. Since V(max) of ATP hydrolysis is attained when three catalytic sites of F1 are saturated, the catalytic site that binds ATP must be closing as the catalytic site that releases products is opening.