A MECHANISM OF PROTON TRANSLOCATION BY F1F0 ATP SYNTHASES SUGGESTED BY DOUBLE MUTANTS OF THE ALPHA-SUBUNIT

Three amino acid residues in the alpha subunit of the Escherichia coli F1F0 ATP synthase are essential for pro ton translocation: Arg(210), Glu(219), and His(245). In this study, the essential glutamic acid has been relocated to position 252 with retention of function. It had been known that Gln(252) can be replaced by Glu without significant effect. To test whether Q252E would function in the absence of Glu(219), a ''site-directed second-site suppressor'' experiment was designed. Saturation mutagenesis was applied to residue Glu(219), and 14 different amino acid substitutions were isolated, five of which permitted growth on succinate minimal medium at 37 degrees C: Asp, Lys, Gly, Ala, and Ser. These results indicate that Q252E can provide the essential carboxyl group normally provided by Glu(219), but that strict requirements are placed on the residue at position 219. We interpret these results to mean that the Q252E must occupy, at least partially, the normal position of Glu(219). We present a novel mechanism of proton translocation by F1F0 ATP synthases that includes a rotating oligomer of c subunits, in which the Asp(61) of two c subunits simultaneously interact with Glu(219) and Arg(210) of the alpha subunit. This mechanism can be adapted for both mitochondrial and sodium-driven bacterial ATP synthases.