INSERTION SCANNING MUTAGENESIS OF SUBUNIT A OF THE F1F0 ATP SYNTHASE NEAR HIS(245) AND IMPLICATIONS ON GATING OF THE PROTON CHANNEL

Subunit a of the E. coli F1F0 ATP synthase was probed by insertion sca nning mutagenesis in a region between residues Glu(219) and His(245). A series of single amino acid insertions, of both alanine and aspartic acid, were constructed after the following residues: 225, 229, 233, 2 38, 243, and 245. The mutants were tested for growth yield, binding of F-1 to membranes, dicyclohexylcarbodiimide sensitivity of ATPase acti vity, ATP-driven proton translocation, and passive proton permeability of membranes stripped of F-1. Significant loss of function was seen o nly with insertions after positions 238 and 243. In contrast, both ins ertions after residue 225 and the alanine insertion after residue 245 were nearly identical in function to the wild type. The other insertio ns showed an intermediate loss of function. Missense mutations of His( 245) to serine and cysteine were nonfunctional, while the W241C mutant showed nearly normal ATPase function. Replacement of Leu(162) by hist idine failed to suppress the 245 mutants, but chemical rescue of H245S was partially successful using acetate, An interaction between Trp(24 1) and His(245) may be involved in gating a ''half-channel'' from the periplasmic surface of F-0 to Asp(61) of subunit a.