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

Abstract

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 aLys 180 and aGly 212 residues that are predicted to be in the proton uptake pathway of the a-subunit were specifically implicated in pHdependent 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 nonfermentative growth and OXPHOS at high pH.Aerobic organisms maximize catabolic energy conservation by carrying out OXPHOS.1 Energy stored in NADH or FADH 2 during catabolism is used to produce a bulk ⌬p, acid and positive out, across the mitochondrial or bacterial cell membrane by respiration-dependent proton extrusion. Inward proton flux through the proton-coupled ATP synthase, energized by the ⌬p, then leads to ATP production (1-4). Among the important unresolved issues is whether protons are always captured from the bulk medium as in Mitchell's (1) chemiosmotic model or whether they can be sequestered as they emerge from the respiratory chain (2, 5-9). Robust H ϩ -coupled OXPHOS by extremely alkaliphilic Bacillus strains growing on non-fermentable carbon sources at external pH values Ն10.5 poses one of the most striking challenges to the strictly bulk energization model (10 -13). At such pH values, maintenance of a cytoplasmic pH that is much lower than the external pH, i.e. a ⌬pH that is acid in, lowers the total chemiosmotic driving force, and yet OXPHOS proceeds optimally (10, 13).A variety of solutions to the energetic conundrum of alkaliphile OXPHOS have been proposed (for reviews, see Refs. 10 and 14). We have hypothesized that special properties of the alkaliphile ATP synthase are needed for OXPHOS at high pH that depend upon the presence of specific amino acid residues or stretches of amino acids in functionally important regions of the membrane-embedded a-and c-subunits of the enzyme (10, 15). Other hypotheses have suggested global features of the alkaliphile membrane, membrane surface, or cell wall-associated polymers (see Refs. 10 and 14) that contribute to the resolution of the alkaliphile energetic problem. It has recently been proposed that a single global feature of this kind, i.e. a sufficiently low pH near the membrane surface, can completely account for alkaliphile OXPHOS (14), ...

Interaction between Cytochrome caa3 and F1F0-ATP Synthase of Alkaliphilic Bacillus pseudofirmus OF4 Is Demonstrated by Saturation Transfer Electron Paramagnetic Resonance and Differential Scanning Calorimetry Assays

“…This will make it possible to test whether one or more of the alkaliphile-specific features of the ATP synthase and cytochrome caa 3 facilitate the interactions observed here and/or apparent proton sequestration in bioenergetic experiments. A candidate in the alkaliphile ATP synthase would be the unusually polar loop on the external side of the a-subunit, near the putative proton uptake pathway, that was shown to be required for optimal OXPHOS at high pH but not critically involved in the proton-gating function (15). A candidate of interest in the cytochrome caa 3 is the unusually acidic patch that was noted when the operon encoding the complex was first cloned and sequenced (17).…”

“…This property reflects a blockage of proton flux both to and from the bulk phase at pH ≥ 9.5 that protects against cytoplasmic alkalinization during sudden exposure to high pH. Two residues of the F 0 -ATP synthase asubunit have been shown to participate in this pH-dependent proton "gating", one of which is also required for ATP synthesis at high pH (15). These findings led us to propose that a kinetically sequestered proton path from the respiratory chain to the ATP synthase at high pH is supported by properties of the participating complexes as well as the membrane and may involve dynamic protein-protein interactions between the ATP synthase and one of the terminal oxidases of the respiratory chain, the proton-pumping caa 3 oxidase.…”

“…This capacity for OXPHOS at pH 10.5 depends upon alkaliphile-specific sequence features of the F 0 -ATP synthase. Mutation of two of these features to the Bacillus consensus sequence resulted in a fully functional enzyme that supported OXPHOS at pH 7.5 but not at pH 10.5 (15). Second, large artificially imposed diffusion potentials fail to energize ATP synthesis above pH 9.5, where non-fermentative growth and respiration-dependent OXPHOS are optimal (13).…”

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

“…1A). Mutagenesis studies of Lys-180 of B. pseudofirmus OF4 showed that its mutation to the consensus glycine of non-alkaliphiles resulted in a major deficit in ATP synthesis at pH 10.5, while significant capacity for ATP synthesis was retained at near neutral pH (37). 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.…”

“…B. pseudofirmus OF4 and other alkaliphilic Bacillus species share sequence motifs in the a-and c-subunits of their proton-coupled ATP synthases (30,33,35). Initial mutagenesis work showed that several of these sequence deviations from the neutralophilic Bacillus consensus have indispensible roles in the synthetic function of the enzyme and non-fermentative growth on malate at high pH, but are not required for hydrolytic ATPase activity or non-fermentative growth of B. pseudofirmus OF4 near neutral pH (36,37). Recent structural studies on the B. pseudofirmus OF4 tri-decameric c-rotor have begun to provide insights into how the major motifs of the alkaliphile c-subunits impact the structure and support the function of the rotor at high pH (31).…”