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J. Biol. Chem., Vol. 282, Issue 24, 17395-17404, June 15, 2007

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A Specific Adaptation in the a Subunit of Thermoalkaliphilic F₁F_O-ATP Synthase Enables ATP Synthesis at High pH but Not at Neutral pH Values^*

Duncan G. G. McMillan¹, Stefanie Keis¹, Peter Dimroth², and Gregory M. Cook³

From the Department of Microbiology and Immunology, Otago School of Medical Sciences, University of Otago, Dunedin, New Zealand and the Institut für Mikrobiologie, ETH-Hönggerberg, CH-8093 Zürich, Switzerland

Analysis of the atp operon from the thermoalkaliphilic Bacillus sp. TA2.A1 and comparison with other atp operons from alkaliphilic bacteria reveals the presence of a conserved lysine residue at position 180 (Bacillus sp. TA2.A1 numbering) within the a subunit of these F₁F_o-ATP synthases. We hypothesize that the basic nature of this residue is ideally suited to capture protons from the bulk phase at high pH. To test this hypothesis, a heterologous expression system for the ATP synthase from Bacillus sp. TA2.A1 (TA2F₁F_o) was developed in Escherichia coli DK8 (atp). Amino acid substitutions were made in the a subunit of TA2F₁F_o at position 180. Lysine (aK180) was substituted for the basic residues histidine (aK180H) or arginine (aK180R), and the uncharged residue glycine (aK180G). ATP synthesis experiments were performed in ADP plus P_i-loaded right-side-out membrane vesicles energized by ascorbate-phenazine methosulfate. When these enzyme complexes were examined for their ability to perform ATP synthesis over the pH range from 7.0 to 10.0, TA2F₁F_o and aK180R showed a similar pH profile having optimum ATP synthesis rates at pH 9.0–9.5 with no measurable ATP synthesis at pH 7.5. Conversely, aK180H and aK180G showed maximal ATP synthesis at pH values 8.0 and 7.5, respectively. ATP synthesis under these conditions for all enzyme forms was sensitive to DCCD. These data strongly imply that amino acid residue Lys¹⁸⁰ is a specific adaptation within the a subunit of TA2F₁F_o to facilitate proton capture at high pH. At pH values near the pK_a of Lys¹⁸⁰, the trapped protons readily dissociate to reach the subunit c binding sites, but this dissociation is impeded at neutral pH values causing either a blocking of the proposed H⁺ channel and/or mechanism of proton translocation, and hence ATP synthesis is inhibited.

Received for publication, December 21, 2006 , and in revised form, March 12, 2007.

^* This work was supported in part by a Marsden grant from the Royal Society of New Zealand (to D. G. G. M. and S. K.). 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.

¹ These authors contributed equally to the experimental work described in this report.

² Supported by the Swiss National Science Foundation and Research Commission of ETH Zürich, Switzerland.

³ To whom correspondence should be addressed: Dept. of Microbiology and Immunology, Otago School of Medical Sciences, University of Otago, P.O. Box 56, Dunedin, New Zealand. Tel.: 64-3-4797722; Fax: 64-3-4798540; E-mail: greg.cook{at}stonebow.otago.ac.nz.

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