A Specific Adaptation in the a Subunit of Thermoalkaliphilic F1FO-ATP Synthase Enables ATP Synthesis at High pH but Not at Neutral pH Values

Duncan G.G Mcmillan,Stefanie Keis,Peter Dimroth,Gregory M Cook

doi:10.1074/jbc.m611709200

Abstract

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 F(1)F(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 (TA2F(1)F(o)) was developed in Escherichia coli DK8 (Deltaatp). Amino acid substitutions were made in the a subunit of TA2F(1)F(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, TA2F(1)F(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 Lys(180) is a specific adaptation within the a subunit of TA2F(1)F(o) to facilitate proton capture at high pH. At pH values near the pK(a) of Lys(180), 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.

Highlights

In studying the role of these residues in growth of the facultative alkaliphile B. pseudofirmus OF4 on fermentable and non-fermentable carbon sources at pH 7.5 and pH 10.5 (31), the authors demonstrate that when lysine residue at position 180 (Lys180) is substituted for glycine, B. pseudofirmus OF4 is no longer able to grow at high pH (i.e. 10.5) on non-fermentable malate
Various amino acid substitutions in the a subunit were introduced to replace the lysine at position 180 and the effect of external pH on ATP synthesis was studied in ADP plus Pi-loaded right-sideout membrane vesicles of E. coli DK8 expressing TA2F1Fo
The ATP synthases from bacteria, mitochondria, and chloroplasts are highly conserved enzymes that have evolved to capture and translocate protons or sodium ions from the bulk phase to drive rotation of an oligomeric c-ring coupled to the synthesis of ATP at the catalytic ␤ subunits

Summary

Thermoalkaliphilic ATP Synthase Adaptation to High pH

On a mechanism with a delocalized ⌬␮Hϩ in the bulk phase but could be overcome by a more localized proton pathway between the respiratory chain and the ATP synthase Support for such a model has been provided by Guffanti et al (22, 23) who demonstrated in Bacillus firmus RAB that a respirationderived ⌬␮Hϩ could drive ATP synthesis at pH 9.0, but a valinomycin-mediated potassium-diffusion potential could not. In the a subunits of alkaliphilic ATP synthases (Fig. 1), an invariant lysine residue at position 180 (Lys180) (Bacillus pseudofirmus OF4 numbering) is located in transmembrane helix 4 (aTMH4) (24, 25). TA2.A1 numbering) (Fig. 1) and we hypothesize that its side chain amino group acts as a base to capture protons at high environmental pH In this communication, we report on the development of a heterologous expression system to overproduce and purify recombinant F1Fo-ATP synthase from Bacillus sp. Various amino acid substitutions in the a subunit were introduced to replace the lysine at position 180 (viz. glycine, histidine, and arginine) and the effect of external pH on ATP synthesis was studied in ADP plus Pi-loaded right-sideout membrane vesicles of E. coli DK8 expressing TA2F1Fo

EXPERIMENTAL PROCEDURES

RESULTS

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DISCUSSION