A tridecameric c ring of the adenosine triphosphate (ATP) synthase from the thermoalkaliphilic Bacillus sp. strain TA2.A1 facilitates ATP synthesis at low electrochemical proton potential

Abstract

SummaryDespite the thermodynamic problem imposed on alkaliphilic bacteria of synthesizing adenosine triphosphate (ATP) against a large inverted pH gradient and consequently a low electrochemical proton potential, these bacteria still utilize a proton-coupled F1Fo-ATP synthase to synthesize ATP. One potential solution to this apparent thermodynamic problem would be the operation of a larger oligomeric c ring, which would raise the ion to ATP ratio, thus facilitating the conversion of a low electrochemical potential into a significant phosphorylation potential. To address this hypothesis, we have purified the oligomeric c ring from the thermoalkaliphilic bacterium Bacillus sp. strain TA2.A1 and determined the number of c-subunits using a novel mass spectrometry method, termed 'laser-induced liquid bead ion desorption' (LILBID). This technique allows the mass determination of non-covalently assembled, detergent-solubilized membrane protein complexes, and hence enables an accurate determination of c ring stoichiometries. We show that the Bacillus sp. strain TA2.A1 ATP synthase harbours a tridecameric c ring. The operation of a c ring with 13 subunits renders the thermodynamic problem of ATP synthesis at alkaline pH less severe and may represent a strategy for ATP synthesis at low electrochemical potential.

The c-ring stoichiometry of ATP synthase is adapted to cell physiological requirements of alkaliphilic Bacillus pseudofirmus OF4

“…This alteration was proposed to enhance both the c-ring stoichiometry and its size in alkaliphilic Bacillus species (19,20,26). Our observations here concur with the expectation that a higher c-ring stoichiometry (i.e, increased i) would be advantageous at a low overall pmf in alkaliphiles (21,32), because the phosphorylation potential for ATP synthesis (ΔG p ) at thermodynamic equilibrium equals i × pmf. The common theme of the enlarged c-ring stoichiometries found in alkaliphilic Bacillus species (c 13 ) and the contrasting smaller c 10 rings found in neutralophilic Bacillus species [e.g., Bacillus PS3 (33), Geobacillus kaustophilus ( Fig.…”

“…However, alkaliphilic bacteria have also developed several adaptation strategies, which in combination support robust growth at high pH (12,13,17,18). These adaptations include alkaliphile-specific features of the ATP synthase, including changes at the c-ring's ion-binding site (19) as well as its size and stoichiometry (19)(20)(21). The N-terminal α-helix of the c-subunits contains a typical membrane protein-packing motif (22) of repetitive glycine residues (GxGxGxG) (Fig.…”

Engineering rotor ring stoichiometries in the ATP synthase

“…A few c-rings in these images were seen to lack individual c-subunits; however, instead of closing this gap so as to form smaller c-rings, these incomplete oligomers have the same shape and diameter as the complete ones (10,25). Furthermore, c-subunits from I. tartaricus and Bacillus TA2.A1 expressed in E. coli were found to assemble correctly into c 11 and c 13 rings, respectively (20,23,24,48), despite the preferred c 10 stoichiometry of the native E. coli c-ring (46). The c-ring sizes also are independent of external factors such as medium pH, host protein expression (48) or host membrane composition (23,24), the source of carbon used by the cell (48,49), and, importantly, the rate of ATP synthesis itself (50,51).…”

“…Furthermore, c-subunits from I. tartaricus and Bacillus TA2.A1 expressed in E. coli were found to assemble correctly into c 11 and c 13 rings, respectively (20,23,24,48), despite the preferred c 10 stoichiometry of the native E. coli c-ring (46). The c-ring sizes also are independent of external factors such as medium pH, host protein expression (48) or host membrane composition (23,24), the source of carbon used by the cell (48,49), and, importantly, the rate of ATP synthesis itself (50,51). Instead, the results presented in this and previous work consistently demonstrate that the c-ring stoichiometries are determined and constrained by the primary structure of the c-subunits (25,52).…”

Biophysical comparison of ATP synthesis mechanisms shows a kinetic advantage for the rotary process

“…synthases and n ≤ maxðΔG ATP =pmfÞ for H + pumps, as previously suggested for chloroplast (57) and mitochondrial (48) ATP synthase. Beyond that, the actual stoichiometry may be determined by the energy available to drive synthesis or pumping versus the importance of rapidly synthesizing ATP or pumping protons.…”