Assembled F1-(alpha beta ) and Hybrid F1-alpha 3beta 3gamma -ATPases from Rhodospirillum rubrum alpha, wild type or mutant beta, and chloroplast gamma subunits. Demonstration of Mg2+versus Ca2+-induced differences in catalytic site structure and function.

Refolding together the expressed alpha and beta subunits of the Rhodospirillum rubrum F(1)(RF(1))-ATPase led to assembly of only alpha(1)beta(1) dimers, showing a stable low MgATPase activity. When incubated in the presence of AlCl(3), NaF and either MgAD(T)P or CaAD(T)P, all dimers associated into closed alpha(3)beta(3) hexamers, which also gained a low CaATPase activity. Both hexamer ATPase activities exhibited identical rates and properties to the open dimer MgATPase. These results indicate that: a) the hexamer, as the dimer, has no catalytic cooperativity; b) aluminium fluoride does not inhibit their MgATPase activity; and c) it does enable the assembly of RrF(1)-alpha(3)beta(3) hexamers by stabilizing their noncatalytic alpha/beta interfaces. Refolding of the RrF(1)-alpha and beta subunits together with the spinach chloroplast F(1) (CF(1))-gamma enabled a simple one-step assembly of two different hybrid RrF(1)-alpha(3)beta(3)/CF(1)gamma complexes, containing either wild type RrF(1)-beta or the catalytic site mutant RrF(1)beta-T159S. They exhibited over 100-fold higher CaATPase and MgATPase activities than the stabilized hexamers and showed very different catalytic properties. The hybrid wild type MgATPase activity was, as that of RrF(1) and CF(1) and unlike its higher CaATPase activity, regulated by excess free Mg(2+) ions, stimulated by sulfite, and inhibited by azide. The hybrid mutant had on the other hand a low CaATPase but an exceptionally high MgATPase activity, which was much less sensitive to the specific MgATPase effectors. All these very different ATPase activities were regulated by thiol modulation of the hybrid unique CF(1)-gamma disulfide bond. These hybrid complexes can provide information on the as yet unknown factors that couple ATP binding and hydrolysis to both thiol modulation and rotational motion of their CF(1)-gamma subunit.