CaATP inhibition of the MgATP-dependent proton pump (H+-ATPase) in bacterial photosynthetic membranes with a mechanism of alternative substrate inhibition

The membrane-bound F1 sector of the H+- ATPase complex (F-type ATPase) in dark-adapted photosynthetic chromatophores is endowed with MgATP- and CaATP-dependent ATPase activities, both sensitive to inhibitors such as oligomycin and venturicidin. Because of contatamination of free Mg2+ and Ca2+ ions in chromatophore preparations, kinetic char acterization of the two hydrolitic reactions can be performed only in the presence of both substrates, using a model for two alternative substrates. The two activities are characterized by similar maximal rates and affinity constants [V-MgATP and V-CaATP: 13 +/- 1 and 10 +/- 1 nmol s(-1) ATP hydrolyzed (mu mol BChl)(-1); K-MgATP and K-CaATP: 0.22 +/- 0.06 and 0.20 +/- 0.05 mM]. However, only the MgATP-dependent ATPase is coupled to Delta<(mu)over tilde>(H+) generation. In this process CaATP acts as an alternative substrate and a competitive inhibitor of the proton pump, with a K-I coincident with K-CaATP for the hydrolytic activity. This finding highlights the central role that the coordination chemistry of the ion-nucleotide complex plays in determining the proton gating mechanism at the catalytic site(s) of the enzyme complex. These results are discussed on the basis of the coordination properties of the ions and of the available information on the protein structure.