Negatively charged residue αAsp-350 of the highly conserved VISIT-DG sequence is required for Pi binding and maintenance of the phosphate-binding subdomain in the catalytic sites of Escherichia coli FF ATP synthase. αAsp-350 is situated in close proximity, 2.88 Å and 3.5 Å, to the conserved known phosphate-binding residues αR376 and βR182. αD350 is also in close proximity, 1.3 Å, to another functionally important residue αG351. Mutation of αAsp-350 to Ala, Gln, or Arg resulted in substantial loss of oxidative phosphorylation and reduction in ATPase activity by 6- to 16-fold. The loss of the acidic side chain in the form of αD350A, αD350Q, and αD350R caused loss of Pi binding. While removal of Arg in the form of αR376D resulted in the loss of Pi binding, the addition of Arg in the form of αG351R did not affect Pi binding. Our data demonstrates that αD350R helps in the proper orientation of αR376 and βR182 for Pi binding. Fluoroaluminate, fluoroscandium, and sodium azide caused almost complete inhibition of wild type enzyme and caused variable inhibition of αD350 mutant enzymes. NBD-Cl (4-chloro-7-nitrobenzo-2-oxa-1, 3-diazole) caused complete inhibition of wild type enzyme while some residual activity was left in mutant enzymes. Inhibition characteristics supported the conclusion that NBD-Cl reacts in βE (empty) catalytic sites. Phosphate protected against NBD-Cl inhibition of wild type and αG351R mutant enzymes but not inhibition of αD350A, αD350Q, αD350R, or αR376D mutant enzymes. These results demonstrate that αAsp-350 is an essential residue required for phosphate binding, through its interaction with αR376 and βR182, for normal function of phosphate binding subdomain and for transition state stabilization in ATP synthase catalytic sites.