A complex of gamma, epsilon, and c subunits rotates in ATP synthase (F0F1) coupling with proton transport. Replacement of beta Ser174 by Phe in beta-sheet4 of the beta subunit (beta S174F) caused slow gamma subunit revolution of the F, sector, consistent with the decreased ATPase activity [M. Nakanishi-Matsui, S. Kashiwagi, T. Ubukata, A. Iwamoto-Kihara, Y. Wada, M. Futai, Rotational catalysis of Escherichia coli ATP synthase F1 sector. Stochastic fluctuation and a key domain of the P subunit, J. Biol. Chem. 282 (2007) 20698-20704]. Modeling of the domain including beta-sheet4 and alpha-helixB predicted that the mutant beta Phe174 residue undergoes strong and weak hydrophobic interactions with beta Ile163 and beta Ile166, respectively. Supporting this prediction, the replacement of beta Ile163 in alpha-helixB by Ala partially suppressed the beta S174F mutation: in the double mutant, the revolution speed and ATPase activity recovered to about half of the levels in the wild-type. Replacement of beta Ile166 by Ala lowered the revolution speed and ATPase activity to the same levels as in beta S174F. Consistent with the weak hydrophobic interaction, beta Ile166 to Ala mutation did not suppress beta S174F. Importance of the hinge domain [phosphatebinding loop (P-loop)/alpha-helixB/loop/beta-sheet4, beta Phe148-beta Gly186] as to driving rotational catalysis is discussed. (c) 2007 Elsevier Inc. All rights reserved.