Intragenic and intergenic suppression of the Escherichia coli ATP synthasesubunit a mutation of Gly-213 to Asn: functional interactions between residues in the proton transport site

Subunit a of the ATP synthase F-o sector contains a transmembrane helix tha t interacts with subunit c and is critical for H+ transport activity. From a cysteine scan in the region around the essential subunit a residue, Arg-2 10, we found that the replacement of aGly-213 greatly attenuated ATP hydrol ysis, ATP-dependent proton pumping and Delta mu(H)-dependent ATP synthesis. Various amino acid substitutions caused similar effects, suggesting that f unctional perturbations were caused by altering the environment or conforma tion of aArg-210. aG213N, which was particularly severe in effect, was supp ressed by two second-site mutations, aL251V and cD61E. These mutations rest ored efficient coupling; the latter also increased ATP-dependent proton tra nsport rates. These results were consistent with the proposed functional in teraction between aArg-210 and cAsp-61, the likely carrier of the transport ed proton. From Arrhenius analysis of steady-state ATP hydrolytic activity, the transport mutants had large increases in the transition-state enthalpi c and entropic parameters. Linear isokinetic relationships demonstrate that the transport mechanism is coupled to the rate-limiting catalytic transiti on-state step, which we have previously shown to involve the rotation of th e gamma subunit in multi-site, co-operative catalysis.