Demonstration of two binding sites for ADP on the isolated β‐subunit of the Rhodospirillum rubrum R₁F₀F₁‐ATP synthase

Abstract

Two ADP binding sites have been demonstrated on the reconstitutively active β‐subunit, that was removed from the Rhodospirillum rubrum membrane‐bound ATP synthase. One is a high affinity site (K d = 0.7 μM) that does not require MgCl2 and is unaffected by it. The second is a low affinity binding site (K d = 80 μM) that is absolutely dependent on MgCl2. For stable binding of ADP to this site, MgCl2 must be present not only during the binding step but also during the elution‐centrifugation step used to separate the β‐subunit bound [3H]ADP from the free ligand. When MgCl2 is removed together with the free ligand [3H]ADP dissociates very rapidly from this second binding site.

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.

“…In light of these results we propose that the low-affinity Mg-dependent nucleotide binding site identified on the isolated R. rubrum /3 subunit (23,24) is the catalytic site of the RrF0-F1 ATP synthase. This site is functional in both ATP synthesis and hydrolysis, in light of our earlier observations on the similar response of both processes in R. rubrum chromatophores to various treatments (21,25,26,31).…”

“…The first is a high-affinity site with a Kd of 5 ,uM for ATP and 9 ,uM for ADP (Table 1), which has been shown to be independent of MgCl2 (23). The second is a low-affinity site with a Kd of 200 ,uM for ATP and 90 ,uM for ADP (Table 1), which has been shown to be absolutely dependent on the presence of MgCl2 (23,24). Modification of the native R. rubrum p subunit by DEPC resulted in complete inhibition of the binding of Pi (Fig.…”

“…These studies have identified one nucleotide binding site on the a subunit (22), whereas the p subunit contained two binding sites for ATP (23) or ADP (24) and one binding site for Pi (41). One of the nucleotide binding sites on the p subunit is a Mg-dependent high-affinity site, which is very similar to the binding site located on the a subunit.…”

“…Direct binding studies with labeled substrates have been carried out only with the a subunit of E. coli (22) and the 8 subunit of R. rubrum (23,24,41). These studies have identified one nucleotide binding site on the a subunit (22), whereas the p subunit contained two binding sites for ATP (23) or ADP (24) and one binding site for Pi (41).…”

“…Such subunits have been obtained so far only from three bacterial sources: a thermophilic bacterium (19), Escherichia coli (20), and Rhodospirillum rubrum (21). They are indeed a much simpler system for study as they show no subunit-subunit interactions and no catalytic activity by themselves, although they can restore ATP synthesis or hydrolysis (or both) to preparations that lack them (19-21).Direct binding studies with labeled substrates have been carried out only with the a subunit of E. coli (22) and the 8 subunit of R. rubrum (23,24,41). These studies have identified one nucleotide binding site on the a subunit (22), whereas the p subunit contained two binding sites for ATP (23) or ADP (24) and one binding site for Pi (41).…”