Rotational Catalysis of Escherichia coli ATP Synthase F1 Sector

• Published in: The Journal of biological chemistry 2007-07, Vol.282 (28), p.20698-20704
• Main Authors: Nakanishi-Matsui, Mayumi, Kashiwagi, Sachiko, Ubukata, Toshiharu, Iwamoto-Kihara, Atsuko, Wada, Yoh, Futai, Masamitsu
• Format: Article
• Language: English
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.M700551200

A complex of γ, ∊, and c subunits rotates in ATP synthase (FoF1) coupled with proton transport. A gold bead connected to the γ subunit of the Escherichia coli F1 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 β subunit key region; consistent with its bulk phase ATPase activities, F1 with the Ser-174 to Phe substitution (βS174F) 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 ∼30-fold higher than the Km value for ATP. A βGly-149 to Ala substitution in the phosphate binding P-loop suppressed the defect of βS174F. The revertant (βG149A/βS174F) 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 βG149A/βS174L. These results indicate that the domain between β-sheet 4 (βSer-174) and P-loop (βGly-149) is important to drive rotation.