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J. Biol. Chem., Vol. 279, Issue 16, 16272-16277, April 16, 2004

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Inverse Regulation of Rotation of F₁-ATPase by the Mutation at the Regulatory Region on the Subunit of Chloroplast ATP Synthase^*

Hanayo Ueoka-Nakanishi, Yoichi Nakanishi¶, Hiroki Konno, Ken Motohashi, Dirk Bald||, and Toru Hisabori**

From the ATP System Project, Exploratory Research for Advanced Technology (ERATO), Japan Science and Technology Agency (JST), 5800-3 Nagatsuta-cho, Midori-ku, Yokohama 226-0026, Japan, The Chemical Resources Laboratory, Tokyo Institute of Technology, Nagatsuta 4259, Midori-ku, Yokohama 226-8503, Japan, ^¶Laboratory of Cell Dynamics, Graduate School of Bioagricultural Sciences, Nagoya University, Nagoya 464-8601, Japan, and ^||Department of Structural Biology, Free University of Amsterdam, De Boelelaan 1087, 1081 Amsterdam, The Netherlands

In F₁-ATPase, the rotation of the central axis subunit relative to the surrounding ₃₃ subunits is coupled to ATP hydrolysis. We previously reported that the introduced regulatory region of the subunit of chloroplast F₁-ATPase can modulate rotation of the subunit of the thermophilic bacterial F₁-ATPase (Bald, D., Noji, H., Yoshida, M., Hirono-Hara, Y., and Hisabori, T. (2001) J. Biol. Chem. 276, 39505–39507). The attenuated enzyme activity of this chimeric enzyme under oxidizing conditions was characterized by frequent and long pauses of rotation of . In this study, we report an inverse regulation of the subunit rotation in the newly engineered F₁-chimeric complex whose three negatively charged residues Glu²¹⁰-Asp²¹¹-Glu²¹² adjacent to two cysteine residues of the regulatory region derived from chloroplast F₁-ATPase were deleted. ATP hydrolysis activity of the mutant complex was stimulated up to 2-fold by the formation of the disulfide bond at the regulatory region by oxidation. We successfully observed inverse redox switching of rotation of using this mutant complex. The complex exhibited long and frequent pauses in its rotation when reduced, but the rotation rates between pauses remained unaltered. Hence, the suppression or activation of the redox-sensitive F₁-ATPase can be explained in terms of the change in the rotation behavior at a single molecule level. These results obtained by the single molecule analysis of the redox regulation provide further insights into the regulation mechanism of the rotary enzyme.

Received for publication, January 20, 2004 , and in revised form, January 26, 2004.

^* This work was supported by the ATP System Project, ERATO funded by the Japan Science and Technology Agency, and in part by the Grant-in-aid for Science Research 13440238 (to T. H.) from Japan Society for the Promotion of 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.

^** To whom correspondence should be addressed. E-mail: thisabor{at}res.titech.ac.jp.

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