Modulation of charge in the phosphate binding site of Escherichia coli ATP synthase

This paper presents a study of the role of positive charge in the P-i binding site of Escherichia coli ATP synthase, the enzyme responsible for ATP-driven proton extrusion and ATP synthesis by oxidative phosphorylation. Arginine residues are known to occur with high propensity in P-i binding sites of proteins generally and in the P-i binding site of the beta E catalytic site of ATP synthase specifically. Removal of natural beta Arg-246 (beta R246A mutant) abrogates P-i binding; restoration of P-i binding was achieved by mutagenesis of either residue beta Asn-243 or alpha Phe-291 to Arg. Both residues are located in the P-i binding site close to beta Arg-246 in x-ray structures. Insertion of one extra Arg at beta-243 or alpha-291 in presence of beta Arg-246 retained P-i binding, but insertion of two extra Arg, at both positions simultaneously, abrogated it. Transition state stabilization was measured using phosphate analogs fluoroaluminate and fluoroscandium. Removal of beta Arg-246 in beta R246A caused almost complete loss of transition state stabilization, but partial rescue was achieved in beta N243R/beta R246A and alpha F291R/beta R246A. beta Arg-243 or alpha Arg-291 in presence of beta Arg-246 was less effective; the combination of alpha F291R/beta N243R with natural beta Arg-246 was just as detrimental as beta R246A. The data demonstrate that electrostatic interaction is an important component of initial P-i binding in catalytic site beta E and later at the transition state complex. However, since none of the mutants showed significant function in growth tests, ATP-driven proton pumping, or ATPase activity assays, it is apparent that specific stereochemical interactions of catalytic site Arg residues are paramount.