Evidence that the Mg-dependent low-affinity binding site for ATP and Pi demonstrated on the isolated beta subunit of the F0.F1 ATP synthase is a catalytic site.

Abstract

Binding sites for one Pi and two ATP or ADP molecules have been identified on the isolated, reconstitutively active beta subunit from the Rhodospirillum rubrum F0.F1 ATP synthase. Chemical modification of this beta subunit by the histidine reagent diethyl pyrocarbonate or by the carboxyl group reagent Woodword's reagent K results in complete inhibition of Pi binding to beta. The same reagents inhibit the binding of ATP to a Mg-dependent low-affinity site but not to a Mg-independent high-affinity site on this beta subunit. The binding stoichiometry of ADP to either site is not affected by these modifications. The beta subunit modified by either one of these reagents retains its capacity to rebind to beta-less chromatophores but not its ability to restore their photo-phosphorylation. These results indicate that the low-affinity Pi binding site on beta is located at the binding site of the gamma-phosphate group of ATP in the Mg-dependent low-affinity nucleotide binding site. This site contains histidine and carboxyl group residues, both of which are required for the binding of Pi and of the gamma-phosphate group of ATP. The same residues must also be involved in the capacity of the isolated beta subunit to restore the catalytic activity of the beta-less ATP synthase. It is therefore concluded that the low-affinity Mg-dependent substrate binding site identified on the isolated beta subunit of the R. rubrum F0.F1 ATP synthase is the catalytic site of this enzyme complex.