Functional importance of αIle-346 and αIle-348 in the catalytic sites of Escherichia coli ATP synthase.

We studied the functional role of highly conserved VISIT-DG sequence residues αIle-346 and αIle-348 in the catalytic sites of Escherichia coli F1Fo ATP synthase. αIle-346 is in close proximity, 2.98 and 3.63 Å, to the two known phosphate binding residues αR376 and βR182; αIle-348 is situated within 3.66 Å from βR182. Single or double mutants of both αI346 and αI348 resulted in a variable loss of oxidative phosphorylation and ATPase activity. Azide, fluoroaluminate, and fluoroscandium caused insignificant to significant inhibition of mutants. Whereas the wild-type enzyme was completely inhibited by NBD-Cl (7-chloro-4-nitrobenzo-2-oxa-1, 3-diazole), a variable extent of inhibition was observed for αI346 and αI348 mutants. MgPi protection against NBD-Cl induced inhibition of wild-type, αI346, and αI348 demonstrated that, although strongly conserved, αI346 and αI348 have no direct role in phosphate binding. Insertion of Arginine in the form of αI346R/βR182A, αI346R/αR376A, or αI348R/βR182A was able to compensate for the absence of known phosphate-binding Arginine residues βR182 and αR376. Results also suggest that αIle-346 and αIle-348 seem to have functional importance in upholding the phosphate-binding subdomain and transition state stabilization in the catalytic sites of E. coli ATP synthase.