Evidence that the Mg-dependent low-affinity binding site for ATP and P(i) demonstrated on the isolated β subunit of the F₀^.F₁ ATP synthase is a catalytic site

Binding sites for one P(i) and two ATP or ADP molecules have been identified on the isolated, reconstitutively active β subunit from the Rhodospirillum rubrum F₀^.F₁ ATP synthase. Chemical modification of this β subunit by the histidine reagent diethyl pyrocarbonate or by the carboxyl group reagent Woodword's reagent K results in complete inhibition of P(i) binding to β. 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 β subunit. The binding stoichiometry of ADP to either site is not affected by these modifications. The β subunit modified by either one of these reagents retains its capacity to rebind to β-less chromatophores but not its ability to restore their photophosphorylation. These results indicate that the low-affinity P(i) binding site on β is located at the binding site of the γ-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 P(i) and of the γ-phosphate group of ATP. The same residues must also be involved in the capacity of the isolated β subunit to restore the catalytic activity of the β-less ATP synthase. It is therefore concluded that the low-affinity Mg-dependent substrate binding site identified on the isolated β subunit of the R. rubrum F₀^.F₁ ATP synthase is the catalytic site of this enzyme complex.