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J. Biol. Chem., Vol. 282, Issue 28, 20698-20704, July 13, 2007

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Rotational Catalysis of Escherichia coli ATP Synthase F₁ Sector

STOCHASTIC FLUCTUATION AND A KEY DOMAIN OF THE SUBUNIT^*

Mayumi Nakanishi-Matsui, Sachiko Kashiwagi, Toshiharu Ubukata, Atsuko Iwamoto-Kihara, Yoh Wada^¶, and Masamitsu Futai¹

From the Futai Special Laboratory, Microbial Chemistry Research Center, Microbial Chemistry Research Foundation, Tokyo 141-0021, Japan and Department of Biochemistry, Faculty of Pharmaceutical Sciences, Iwate Medical University, Iwate 028-3694, Japan, Department of Bioscience, Nagahama Institute of Bioscience and Technology, Nagahama, Shiga 526-0829, Japan, and ^¶Department of Biological Sciences, The Institute of Scientific and Industrial Research, Osaka University, Ibaraki, Osaka 567-0041, Japan

A complex of , , and c subunits rotates in ATP synthase (FoF₁) coupled with proton transport. A gold bead connected to the 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 subunit key region; consistent with its bulk phase ATPase activities, F₁ with the Ser-174 to Phe substitution (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 30-fold higher than the K_m value for ATP. A Gly-149 to Ala substitution in the phosphate binding P-loop suppressed the defect of S174F. The revertant (G149A/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 G149A/S174L. These results indicate that the domain between -sheet 4 (Ser-174) and P-loop (Gly-149) is important to drive rotation.

Received for publication, January 19, 2007 , and in revised form, May 16, 2007.

^* This study was supported by CREST, Japan Science and Technology Agency, and the Japanese Ministry of Education, Culture, and Science. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ To whom correspondence should be addressed. Tel.: 81-19-651-5111; Fax: 81-19-698-1843; E-mail: futaim{at}iwate-med.ac.jp.

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