The Trapping of Different Conformations of the Escherichia coli F1 ATPase by Disulfide Bond Formation: EFFECT ON NUCLEOTIDE BINDING AFFINITIES OF THE CATALYTIC SITES

Abstract

Two mutants of the Escherichia coli F1 ATPase, betaY331W:E381C/epsilonS108C and alphaS411C/betaY331W/epsilonS108C, have been used to relate nucleotide binding in catalytic sites with different interactions of the stalk-forming subunits gamma and epsilon at the alpha3beta3 subunit domain. Essentially full yield cross-linking between beta + gamma and beta + epsilon, or between alpha + gamma and alpha + epsilon, was obtained in these mutants by Cu2+-induced disulfide bond formation, thereby trapping the enzyme in states with the small subunits interacting either with beta or alpha subunits. The presence of the Trp for beta Tyr-331 in both mutants allowed direct measurement of nucleotide occupancy of catalytic sites. Before cross-linking, Mg2+ATP could be bound in all three catalytic sites in both mutants with a Kd of around 0.1 microM for the highest affinity site and Kd values of approximately 2 microM and 30-40 microM for the second and third sites, respectively. In the absence of Mg2+, ATP also bound in all three catalytic sites but with a single low affinity (above 100 microM) in both mutants. Cu2+-induced cross-linking of ECF1 from the mutant betaY331W:E381C/epsilonS108C had very little effect on nucleotide binding. The binding affinities of the three catalytic sites for Mg2+ATP were not significantly altered from those obtained before cross-linking, and the enzyme still switched between cooperative binding and equal binding affinities of the three catalytic sites (when Mg2+ was absent). When the gamma and epsilon subunits were cross-linked to alpha subunits, ATP binding in the highest affinity catalytic site was dramatically altered. This site became closed so that nucleotide (ATP or ADP) that had been bound into it prior to cross-linking was trapped and could not exchange out. Also, ATP or ADP could not enter this site, although empty, once the enzyme had been cross-linked. Finally, cross-linking of the gamma and epsilon to the alpha subunits prevented the switching between cooperative binding and the state where the three catalytic sites are equivalent. We argue that the conformation of the enzyme in which the small subunits are at alpha subunits occurs during functioning of the enzyme in the course of the rotation of gamma and epsilon subunits within the alpha3beta3 hexamer and that this may be the activated state for ATP synthesis.