Inverse regulation of rotation of F1-ATPase by the mutation at the regulatory region on the γ subunit of chloroplast ATP synthase

In F-1-ATPase, the rotation of the central axis subunit gamma relative to the surrounding alpha(3)beta(3) subunits is coupled to ATP hydrolysis. We previously reported that the introduced regulatory region of the gamma subunit of chloroplast F-1-ATPase can modulate rotation of the gamma subunit of the thermophilic bacterial F-1-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 gamma. In this study, we report an inverse regulation of the gamma subunit rotation in the newly engineered F-1-chimeric complex whose three negatively charged residues Glu(210)-Asp(211)-Glu(212) adjacent to two cysteine residues of the regulatory region derived from chloroplast F-1-ATPase gamma 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 gamma using this mutant complex. The complex exhibited long and frequent pauses in its gamma rotation when reduced, but the rotation rates between pauses remained unaltered. Hence, the suppression or activation of the redox-sensitive F-1-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.