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

Abstract

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 Pi binding sites of proteins generally and in the Pi binding site of the βE catalytic site of ATP synthase specifically. Removal of natural βArg-246 (βR246A mutant) abrogates Pi binding; restoration of Pi binding was achieved by mutagenesis of either residue βAsn-243 or αPhe-291 to Arg. Both residues are located in the Pi binding site close to βArg-246 in x-ray structures. Insertion of one extra Arg at β-243 or α-291 in presence of βArg-246 retained Pi 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 βArg-246 in βR246A caused almost complete loss of transition state stabilization, but partial rescue was achieved in βN243R/βR246A and αF291R/βR246A. βArg-243 or αArg-291 in presence of βArg-246 was less effective; the combination of αF291R/βN243R with natural βArg-246 was just as detrimental as βR246A. The data demonstrate that electrostatic interaction is an important component of initial Pi binding in catalytic site βE 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. © 2005 by The American Society for Biochemistry and Molecular Biology, Inc.