Rotational catalysis of Escherichia coli ATP synthase F1 sector -: Stochastic fluctuation and a key domain of the β subunit

A complex of gamma, is an element of, and c subunits rotates in ATP synthase (FoF(1)) coupled with proton transport. A gold bead connected to the gamma subunit of the Escherichia coli F-1 sector exhibited stochastic rotation, confirming a previous study (Nakanishi-Matsui, M., Kashiwagi, S., Hosokawa, H., Cipriano, D. J., Dunn, S. D., Wada, Y., and Futai, M. (2006) J. Biol. Chem. 281, 4126 - 4131). A similar approach was taken for mutations in the beta subunit key region; consistent with its bulk phase ATPase activities, F1 with the Ser-174 to Phe substitution (beta S174F) exhibited a slower single revolution time ( time required for 360 degree revolution) and paused almost 10 times longer than the wild type at one of the three 120 positions during the stepped revolution. The pause positions were probably not at the "ATP waiting" dwell but at the "ATP hydrolysis/ product release" dwell, since the ATP concentration used for the assay was similar to 30-fold higher than the Km value for ATP. A beta Gly-149 to Ala substitution in the phosphate binding P-loop suppressed the defect of beta S174F. The revertant (beta G149A/beta S174F) exhibited similar rotation to the wild type, except that it showed long pauses less frequently. Essentially the same results were obtained with the Ser-174 to Leu substitution and the corresponding revertant beta G149A/beta S174L. These results indicate that the domain between beta-sheet 4 (beta Ser-174) and P-loop (beta Gly-149) is important to drive rotation.