ATP Synthesis by F₀F₁-ATP Synthase Independent of Noncatalytic Nucleotide Binding Sites and Insensitive to Azide Inhibition*

Abstract

ATP hydrolyzing activity of a mutant α₃β₃γ subcomplex of F₀F₁-ATP synthase (ΔNC) from the thermophilicBacillus PS3, which lacked noncatalytic nucleotide binding sites, was inactivated completely soon after starting the reaction (Matsui, T., Muneyuki, E., Honda, M., Allison, W. S., Dou, C., and Yoshida, M. (1997) J. Biol. Chem. 272, 8215–8221). This inactivation is caused by rapid accumulation of the “MgADP inhibited form” which, in the case of wild-type enzyme, would be relieved by ATP binding to noncatalytic sites. We reconstituted F₀F₁-ATP synthase into liposomes together with bacteriorhodopsin and measured illumination-driven ATP synthesis. Remarkably, ΔNC F₀F₁-ATP synthase catalyzed continuous turnover of ATP synthesis while it could not promote ATP-driven proton translocation. ATP synthesis by ΔNC F₀F₁-ATP synthase, as well as wild-type enzyme, proceeded even in the presence of azide, an inhibitor of ATP hydrolysis that stabilizes the MgADP inhibited form. The time course of ATP synthesis by ΔNC F₀F₁-ATP synthase was linear, and gradual acceleration to the maximal rate, which was observed for the wild-type enzyme, was not seen. Thus, ATP synthesis can proceed without nucleotide binding to noncatalytic sites even though the rate is sub-maximal. These results indicate that the MgADP inhibited form is not produced in ATP synthesis reaction, and in this regard, ATP synthesis may not be a simple reversal of ATP hydrolysis.