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FoF1-ATP synthase (FoF1) synthesizes ATP in the F1 portion when pro-tons flow through Fo to rotate the shaft common to F1 and Fo. Rotary synthesis in isolated F1 alone has been shown by applying external torque to F1 of thermophilic origin. | IFEBS Journal Efficient ATP synthesis by thermophilic Bacillus FoF1-ATP synthase Naoki Soga1 Kazuhiko Kinosita Jr1 Masasuke Yoshida2 3 and Toshiharu Suzuki2 1 Department of Physics Faculty of Science and Engineering Waseda University Tokyo Japan 2 ATP Synthesis Regulation Project ICORP Japan Science and Technology Agency JST Tokyo Japan 3 Department of Molecular Bioscience Kyoto Sangyo University Kyoto City Japan Keywords ATP synthesis Michaelis-Menten constants reconstitution temperature TFoFi Correspondence K. Kinosita Jr Department of Physics Faculty of Science and Engineering Waseda University 3-4-1 Okubo Shinjuku-ku Tokyo 169-8555 Japan Fax 81 3 5952 5877 Tel 81 3 5952 5871 E-mail kazuhiko@waseda.jp Re-use of this article is permitted in accordance with the Terms and Conditions set out at http wileyonlinelibrary.com onlineopen OnlineOpen_Terms Received 6 March 2011 revised 19 April 2011 accepted 16 May 2011 FqFj-ATP synthase FoFj synthesizes ATP in the F1 portion when protons flow through Fo to rotate the shaft common to F1 and Fo. Rotary synthesis in isolated Fl alone has been shown by applying external torque to Fl of thermophilic origin. Proton-driven ATP synthesis by thermophilic Bacillus PS3 FoF1 TFoF1 however has so far been poor in vitro of the order of 1 s-1 or less hampering reliable characterization. Here by using a mutant TFoF1 lacking an inhibitory segment of the e-subunit we have developed highly reproducible simple procedures for the preparation of active proteoliposomes and for kinetic analysis of ATP synthesis which was driven by acid-base transition and K -diffusion potential. The synthesis activity reached 16 s-1 at 30 C with a Q10 temperature coefficient of 3-4 between 10 and 30 C suggesting a high level of activity at the physiological temperature of 60 C. The Michaelis-Menten constants for the substrates ADP and inorganic phosphate were 13 IM and 0.55 mM respectively which are an order of magnitude lower than previous estimates and are .
