Identification of the betaTP site in the x-ray structure of F1-ATPase as the high-affinity catalytic site.

ATP synthase uses a unique rotary mechanism to couple ATP synthesis and hydrolysis to transmembrane proton translocation. The F(1) subcomplex has three catalytic nucleotide binding sites, one on each beta subunit, with widely differing affinities for MgATP or MgADP. During rotational catalysis, the sites switch their affinities. The affinity of each site is determined by the position of the central gamma subunit. The site with the highest nucleotide binding affinity is catalytically active. From the available x-ray structures, it is not possible to discern the high-affinity site. Using fluorescence resonance energy transfer between tryptophan residues engineered into gamma and trinitrophenyl nucleotide analogs on the catalytic sites, we were able to determine that the high-affinity site is close to the C-terminal helix of gamma, but at considerable distance from its N terminus. Thus, the beta(TP) site in the x-ray structure [Abrahams JP, Leslie AGW, Lutter R, Walker JE (1994) Nature 370:621-628] is the high-affinity site, in agreement with the prediction of Yang et al. [Yang W, Gao YQ, Cui Q, Ma J, Karplus M (2003) Proc Natl Acad Sci USA 100:874-879]. Taking into account the known direction of rotation, the findings establish the sequence of affinities through which each catalytic site cycles during MgATP hydrolysis as low --> high --> medium --> low.