Role of αPhe-291 residue in the phosphate-binding subdomain of catalytic sites of Escherichia coli ATP synthase

The role of alpha Phe-291 residue in phosphate binding by Escherichia coli F1F0-ATP synthase was examined. X-ray structures of bovine mitochondrial enzyme suggest that this residue resides in close proximity to the conserved beta R246 residue. Herein, we show that mutations alpha F291D and alpha F291E in E. coli reduce the ATPase activity of F1F0 membranes by 350-fold. Yet, significant oxidative phosphorylation activity is retained. In contrast to wild-type, ATPase activities of mutants were not inhibited by MgADP-azide, MgADP-fluoroaluminate, or MgADP-fluoroscandium. Whereas, 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole (NBD-Cl) inhibited wild-type ATPase essentially completely, ATPase in mutants was inhibited maximally by similar to 75%, although reaction still occurred at residue beta Tyr-297, proximal to alpha Phe-291 in the phosphate-binding pocket. Inhibition characteristics supported the conclusion that NBD-Cl reacts in PE (empty) catalytic sites, as shown previously by X-ray structure analysis. Phosphate protected against NBD-Cl inhibition in wild-type but not in mutants. In addition, our data suggest that the interaction of alpha Phe-291 with phosphate during ATP hydrolysis or synthesis may be distinct. (C) 2008 Elsevier Inc. All rights reserved.