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J. Biol. Chem., Vol. 279, Issue 25, 26546-26554, June 18, 2004

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Replacement of Amino Acid Sequence Features of a- and c-Subunits of ATP Synthases of Alkaliphilic Bacillus with the Bacillus Consensus Sequence Results in Defective Oxidative Phosphorylation and Non-fermentative Growth at pH 10.5^*

ZhenXiong Wang, David B. Hicks, Arthur A. Guffanti, Katisha Baldwin, and Terry Ann Krulwich

From the Department of Pharmacology and Biological Chemistry, Mount Sinai School of Medicine, New York, New York 10029

Mitchell's (Mitchell, P. (1961) Nature 191, 144–148) chemiosmotic model of energy coupling posits a bulk electrochemical proton gradient (p) as the sole driving force for proton-coupled ATP synthesis via oxidative phosphorylation (OXPHOS) and for other bioenergetic work. Two properties of proton-coupled OXPHOS by alkaliphilic Bacillus species pose a challenge to this tenet: robust ATP synthesis at pH 10.5 that does not correlate with the magnitude of the p and the failure of artificially imposed potentials to substitute for respiration-generated potentials in energizing ATP synthesis at high pH (Krulwich, T. (1995) Mol. Microbiol. 15, 403–410). Here we show that these properties, in alkaliphilic Bacillus pseudofirmus OF4, depend upon alkaliphile-specific features in the proton pathway through the a- and c-subunits of ATP synthase. Site-directed changes were made in six such features to the corresponding sequence in Bacillus megaterium, which reflects the consensus sequence for non-alkaliphilic Bacillus. Five of the six single mutants assembled an active ATPase/ATP synthase, and four of these mutants exhibited a specific defect in non-fermentative growth at high pH. Most of these mutants lost the ability to generate the high phosphorylation potentials at low bulk p that are characteristic of alkaliphiles. The aLys¹⁸⁰ and aGly²¹² residues that are predicted to be in the proton uptake pathway of the a-subunit were specifically implicated in pH-dependent restriction of proton flux through the ATP synthase to and from the bulk phase. The evidence included greatly enhanced ATP synthesis in response to an artificially imposed potential at high pH. The findings demonstrate that the ATP synthase of extreme alkaliphiles has special features that are required for non-fermentative growth and OXPHOS at high pH.

Received for publication, February 3, 2004 , and in revised form, March 11, 2004.

^* This work was supported by National Institutes of Health Research Grant GM28454. 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.

Present address: Dept. of Medical Oncology, Dana Farber Cancer Inst., Harvard Medical School, Boston, MA 02115.

To whom correspondence should be addressed: Dept. of Pharmacology and Biological Chemistry, Box 1603, Mount Sinai School of Medicine, 1 G. Levy Place, New York, NY 10029. E-mail: terry.krulwich{at}mssm.edu.

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