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J. Biol. Chem., Vol. 280, Issue 30, 27981-27989, July 29, 2005

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Modulation of Charge in the Phosphate Binding Site of Escherichia coli ATP Synthase^*

Zulfiqar Ahmad and Alan E. Senior

From the Department of Biochemistry and Biophysics, University of Rochester Medical Center, Rochester, New York 14642

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 E catalytic site of ATP synthase specifically. Removal of natural Arg-246 (R246A mutant) abrogates P_i binding; restoration of P_i binding was achieved by mutagenesis of either residue Asn-243 or Phe-291 to Arg. Both residues are located in the P_i binding site close to Arg-246 in x-ray structures. Insertion of one extra Arg at -243 or -291 in presence of 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 Arg-246 in R246A caused almost complete loss of transition state stabilization, but partial rescue was achieved in N243R/R246A and F291R/R246A. Arg-243 or Arg-291 in presence of Arg-246 was less effective; the combination of F291R/N243R with natural Arg-246 was just as detrimental as R246A. The data demonstrate that electrostatic interaction is an important component of initial P_i binding in catalytic site 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.

Received for publication, April 12, 2005 , and in revised form, June 2, 2005.

^* This work was supported by National Institutes of Health Grant GM25349 (to A. E. S.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

To whom correspondence should be addressed: Dept. of Biochemistry and Biophysics, Box 712, University of Rochester Medical Center, Rochester, NY 14642. Tel.: 585-275-6645; Fax: 585-271-2683; E-mail: alan_senior{at}urmc.rochester.edu.

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