ATP SYNTHESIS BY F0F1-ATP SYNTHASE INDEPENDENT OF NONCATALYTIC NUCLEOTIDE-BINDING SITES AND INSENSITIVE TO AZIDE INHIBITION

ATP hydrolyzing activity of a mutant alpha(3) beta(3) gamma subcomplex of F0F1-ATP synthase (Delta NC) from the thermophilic Bacillus PS3, w hich lacked noncatalytic nucleotide binding sites, was inactivated com pletely soon after starting the reaction (Matsui, T., Muneyuki, E., Ho nda, M., Allison, W. S., Dou, C., and Yoshida, M. (1997) J. Biol. Chem . 272, 8215-8221), This inactivation is caused by rapid accumulation o f the ''MgADP inhibited form'' which, in the case of wild-type enzyme, would be relieved by ATP binding to noncatalytic sites. We reconstitu ted F0F1-ATP synthase into liposomes together with bacteriorhodopsin a nd measured illumination-driven ATP synthesis. Remarkably, Delta NC F0 F1-ATP synthase catalyzed continuous turnover of ATP synthesis while i t could not promote ATP-driven proton translocation. ATP synthesis by Delta NC F0F1-ATP synthase, as well as wild-type enzyme, proceeded eve n in the presence of azide, an inhibitor of ATP hydrolysis that stabil izes the MgADP inhibited form. The time course of ATP synthesis by Del ta NC F0F1-ATP synthase was linear, and gradual acceleration to the ma ximal rate, which was observed for the wild-type enzyme, was not seen. Thus, ATP synthesis can proceed without nucleotide binding to noncata lytic sites even though the rate is sub-maximal. These results indicat e that the MgADP inhibited form is not produced in ATP synthesis react ion, and in this regard, ATP synthesis may not be a simple reversal of ATP hydrolysis.