ATP synthase uses a unique rotary mechanism to couple ATP synthesis and hydrolysis to transmembrane proton translocation. The F1 subcomplex has three catalytic nucleotide binding sites, one on each beta subunit, at the interface to the adjacent alpha subunit. In the x-ray structure of F1 (Abrahams, J. P., Leslie, A. G. W., Lutter, R., and Walker, J. E. (1994) Nature 370, 621-628), the three catalytic beta/alpha interfaces differ in the extent of inter-subunit interactions between the C termini of the beta and alpha subunits. At the closed betaDP/alphaDP interface, a hydrogen-bonding network is formed between both subunits, which is absent at the more open betaTP/alphaTP interface and at the wide open betaE/alphaE interface. The hydrogen-bonding network reaches from betaL328 (Escherichia coli numbering) and betaQ441 via alphaQ399, betaR398, and alphaE402 to betaR394, and ends in a cation/pi interaction between betaR394 and alphaF406. Using mutational analysis in E. coli ATP synthase, the functional importance of the betaDP/alphaDP hydrogen-bonding network is demonstrated. Its elimination results in a severely impaired enzyme but has no pronounced effect on the binding affinities of the catalytic sites. A possible role for the hydrogen-bonding network in coupling of ATP synthesis/hydrolysis and rotation will be discussed.