The Ser-beta 174 residue of the Escherichia coli H+-ATPase beta subunit has been shown to be near the catalytic site together with Gly-beta 149, Gly-beta 172, Glu-beta 192, and Val-beta 198 (Iwamoto, A., Park, M.-Y., Maeda, M., and Futai, M. (1993) J. Biol. Chem. 268, 3156-3160). In this study, we introduced various residues at position 174 and found that the larger the side chain volume of the residue introduced, the lower the enzyme activity became. The Phe-beta 174 mutant was defective in energy coupling between catalysis and transport, whereas the Leu-beta 174 mutant could couple efficiently, although both mutants had essentially the same ATPase activities (similar to 10% of the wild type). The defective energy coupling of the Phe-beta 174 mutant was suppressed by the second mutation (Arg-alpha 296 --> Cys) in the alpha subunit. The Cys-alpha 296/Phe-beta 174 mutant had essentially the same membrane ATPase activity as the Phe-beta 174 single mutant when assayed under the conditions that stabilize the double mutant enzyme. These results indicate the importance of the alpha/beta interaction, especially that between the regions near Arg-alpha 296 and Ser-beta 174, for energy coupling in the H+-ATPase. The 2 residues (Ser-beta 174 and Arg-alpha 296) may be located nearby at the interface of the two subunits. About 1 mol of N-[C-14]ethylmaleimide could bind to 1 mol of the alpha subunit of Cys-alpha 296/Phe-beta 174 or Cys-alpha 296 mutant ATPase, but could not inhibit the enzyme activity. This is the first intersubunit mutation/suppression approach to ATPase catalysis and its energy coupling.