Abstract
In F(1)-ATPase, the smallest known motor enzyme, unidirectional rotation of the central axis subunit gamma is coupled to ATP hydrolysis. In the present study, we report the redox switching of the rotation of this enzyme. For this purpose, the switch region from the gamma subunit of the redox-sensitive chloroplast F(1)-ATPase was introduced into the bacterial F(1)-ATPase. The ATPase activity of the obtained complex was increased up to 3-fold upon reduction (Bald, D., Noji, H., Stumpp, M. T., Yoshida, M. & Hisabori, T. (2000) J. Biol. Chem. 275, 12757-12762). Here, we successfully observed the modulation of rotation of gamma in this chimeric complex by changes in the redox conditions. In addition we revealed that the suppressed enzymatic activity of the oxidized F(1)-ATPase complex was characterized by more frequent long pauses in the rotation of the gamma subunit. These findings obtained by the single molecule analysis therefore provide new insights into the mechanisms of enzyme regulation.
Highlights
From ʈPRESTO, ‡Chemical Resources Laboratory, Tokyo Institute of Technology, Nagatsuta 4259, Midori-ku, Yokohama, Kanagawa 226-8503 and §CREST Genetic Programming Team 13, Teikyo University Biotechnology Research Center 3F, Nogawa 907, Miyamae-ku, Kawasaki, Kanagawa 216-0001, Japan In F1-ATPase, the smallest known motor enzyme, unidirectional rotation of the central axis subunit ␥ is coupled to ATP hydrolysis
The rotation of ␥ in the central cavity of ␣33 was suggested by biochemical experiments [10], and direct visualization of the rotation of an actin filament attached to the ␥ subunit of F1 showed unequivocally that ␥ rotates unidirectionally during ATP hydrolysis [11,12,13,14]
Redox Control of Rotation of ␥—In the present study, the redox control of the activity of the redox-sensitive ␣33␥TCT complex was observed by monitoring the rotation of the ␥ subunit
Summary
In addition we revealed that the suppressed enzymatic activity of the oxidized F1-ATPase complex was characterized by more frequent long pauses in the rotation of the ␥ subunit. This switch region consists of 37 amino acid residues (Pro194–Ile230 in case of the spinach chloroplast enzyme) containing two cysteine residues, but its structure is unknown, and there are no data available on how reduction of the disulfide bond leads to activation of the enzyme.
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