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

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 (⌬atp). 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.For nearly all aerobic life on earth, the F 1 F o -ATP synthases are the major enzymes responsible for providing ATP to drive endergonic reactions of the cell. These two domain membranebound enzymes are found in mitochondria, chloroplasts, and bacteria, coupling protons or Na ϩ ions to the synthesis of ATP (1, 2). The intracellular water-soluble F 1 domain contains the catalytic sites of the enzyme, while the bulk of the hydrophobic F o domain is embedded in the cytoplasmic membrane and contains the functional center for the capture and translocation of protons. The bacterial F 1 domain has a stoichiometry of ␣ 3 ␤ 3 ␥␦⑀ in which the catalytic nucleotide-binding sites are formed by each of the ␤ subunits and the non-catalytic nucleotide-binding sites are located in the ␣ subunits (3-5). The F o domain consists of one a subunit, two b subunits making the stator, and a ring of 10 -15 c subunits depending on the species (6 -12). The coupling of ion translocation to the rotational mechanism of the F o c-ring forces open, loose, or tight conformational changes within the ␤ subunits of the F 1 domain, driving the synthesis of ATP from ADP and inorganic phosphate (13). Conversely, the F 1 domain can h...

The ATP Synthase a-subunit of Extreme Alkaliphiles Is a Distinct Variant

“…This was consistent with their growth phenotypes on malate (Fig. 2B) and with the hypothesis that the positive charge of Lys-180 is essential for ATP synthesis at highly alkaline pH (37,38). By contrast, the K180G and the K180H mutants, and to a lesser extent the K180A mutant, exhibited much more ATP synthesis in the vesicle assay at pH 7.5 than expected from their growth phenotype on malate at pH 7.5 (Fig.…”

“…Robust support of ATP synthesis at pH 7.0 -7.5 was conferred upon the enzyme when histidine or glycine replaced the lysine, whereas replacement of Lys-180 with arginine resulted in robust synthase activity only at pH Ն8.5. They proposed that a synthase with Lys-180 might be unable to synthesize ATP at near neutral or neutral pH (38). The subsequent finding that C. thermarum TA2.A1 could not grow non-fermentatively below pH 8.0 was consistent with that proposal (39).…”

“…Mutations in Other Alkaliphile-specific a-subunit Residues in B. pseudofirmus OF4 That Are Absent from C. thermarum TA2.A1 Reduce Malate Growth at High pH-The complete inability of the B. pseudofirmus OF4 a-subunit mutant with the K180R substitution to support ATP synthesis or malate growth was in sharp contrast to the efficacy of a comparable mutant to support ATP synthesis at alkaline pH by the enzyme from thermoalkaliphilic C. thermarum (38). This increased the paradox noted in the introduction that is posed by the inability of the thermoalkaliphilic enzyme to function synthetically at pH 7.5 unless its native Lys-180 was replaced by glycine or histidine (38).…”

“…If so, it was likely that, if the Lys-180 of B. pseudofirmus OF4 was replaced with the same residues that had been introduced into the thermoalkaliphile synthase, their effects would be very different because of the distinct scaffold into which they were introduced. Here, we studied mutants with changes in the Lys-180 of B. pseudofirmus OF4 that were made in the native atp locus of the alkaliphile chromosome to either alanine, glycine, cysteine, arginine, or histidine, a somewhat larger panel from those made in the C. thermarum TA2.A1 synthase (38). In addition, we constructed A, putative TMH4 and TMH5 are shown for the following organisms, with their accession numbers in parentheses.…”

“…We hypothesized that, at high pH, Lys-180 is a participant of the proton uptake path that is required for capture of entering protons and passage of the protons to the interface of the c-ring with the a-subunit. Subsequently, McMillan et al (38) expressed the full hexahistidine-tagged atp operon from C. thermarum TA2.A1 in an E. coli mutant from which the native atp operon was deleted. They found that this thermoalkaliphile enzyme failed to support non-fermentative growth of E. coli or to carry out in vitro ATP synthesis below pH 8.0.…”