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

Abstract

A lysine residue in the putative proton uptake pathway of the ATP synthase a-subunit is found only in alkaliphilic Bacillus species and is proposed to play roles in proton capture, retention and passage to the synthase rotor. Here, Lys-180 was replaced with alanine (Ala), glycine (Gly), cysteine (Cys), arginine (Arg), or histidine (His) in the chromosome of alkaliphilic Bacillus pseudofirmus OF4. All mutants exhibited octylglucosidestimulated ATPase activity and ␤-subunit levels at least as high as wild-type. Purified mutant F 1 F 0 -ATP synthases all contained substantial a-subunit levels. The mutants exhibited diverse patterns of native (no octylglucoside) ATPase activity and a range of defects in malate growth and in vitro ATP synthesis at pH 10. Proton-coupled F 1 F 0 -ATP synthases are centrally important for non-fermentative cells that energize ATP synthesis using the energy of an electrochemical proton gradient, the PMF, 3 across the cytoplasmic or thylakoid membrane (bacteria) and across the mitochondrial or chloroplast thylakoid membrane (eukaryotes) (1-3). ATP synthases are composed of two domains, with bacterial synthases having simpler structures than eukaryotic homologues. The cytoplasmically located, soluble F 1 domain encompasses three catalytic ␣-and ␤-subunit pairs and single ␥-, ␦-, and ⑀-subunits. The membrane-associated F 0 domain is composed of a single a-subunit, two b-subunits, and multiple c-subunits (2, 4 -6). ATP synthases function as rotary nano-machines, in which inward translocation of protons through the F 0 domain leads to rotation of a membrane-embedded ring-like rotor (2, 3, 7-10). The rotor is formed from 10 -15 hairpin-like c-subunits, depending upon the organism (11-16). Essential steps in coupling of ATP synthesis to the PMF include the protonation of successive c-subunits of the rotor and, after full rotation of a protonated c-subunit, de-protonation of that subunit through interactions of c-subunits of the rotor with the a-subunit stator component (4,5,7). No high resolution structural data are yet available for the a-subunit, but extensive biochemical and genetic evidence indicates that this ATP synthase subunit plays roles in providing the proton path from outside the membrane surface to the carboxylates of interacting c-subunits of the rotor (4, 17-24). An essential, conserved arginine in TMH4 (Arg-210 in Escherichia coli) is proposed to prevent proton short-cutting to the cytoplasm without rotation (25) and to cause a shift in the pK a of the essential carboxylate so that the proton that has completed rotation dissociates and enters the proton exit pathway leading to the cytoplasm. That proton exit pathway is also likely to be within the a-subunit (4, 5, 18, 23, 26 -28).Valuable insights into the mechanism of ATP synthase have been obtained from studies of bacterial synthases because of the ease of introducing and analyzing effects of mutations (8, 14, 15, 29 -31). Our own studies have focused on the ATP synthase of alkaliphilic Bacillus species. The model extreme alka...

Molecular aspects of bacterial pH sensing and homeostasis

“…The ATP synthases of aerobic alkaliphilic Bacillus species function in the synthetic direction. They have specific sequence motifs in proton-translocating subunit- a and subunit- c that support function at high pH and guard against cytoplasmic proton loss during ATP synthesis 134,135,136,137 . Mutations of these motifs to the non-alkaliphile consensus sequence leads to reduced ATP synthase activity, usually with a greater effect at pH 10.5 than at pH 7.5.…”

“…Mutations of these motifs to the non-alkaliphile consensus sequence leads to reduced ATP synthase activity, usually with a greater effect at pH 10.5 than at pH 7.5. The magnitude of the defect in ATP synthase activity correlates with a loss of the mutants’ capacities for pH homeostasis during a sudden alkaline shift in pH out 134,137 . Some ATP synthase motif mutations also lead to proton leakiness 134,136,137 .…”

“…The magnitude of the defect in ATP synthase activity correlates with a loss of the mutants’ capacities for pH homeostasis during a sudden alkaline shift in pH out 134,137 . Some ATP synthase motif mutations also lead to proton leakiness 134,136,137 . Thus the alkaliphile synthase is adapted to promote both function in pH homeostasis and ATP synthesis at high pH.…”

Mutations in a Helix-1 Motif of the ATP Synthase c-Subunit of Bacillus pseudofirmus OF4 Cause Functional Deficits and Changes in the c-Ring Stability and Mobility on Sodium Dodecyl Sulfate–Polyacrylamide Gel Electrophoresis

“…Evidence suggests that such sequestration of protons near the surface may allow protons to reach the proton-coupled F 1 F o -ATP synthase (hereafter referred to as ATP synthase) of alkaliphiles before they fully equilibrate with the bulk medium that can be above pH 11 (1–4). Additionally, successful function of the alkaliphile ATP synthase at high pH depends upon specific adaptations of both the respiratory chain complexes and the ATP synthase (5–9). Adaptations of the ATP synthase to function at alkaline pH were found in the membrane-embedded F o complex (5, 8–11).…”

“…Additionally, successful function of the alkaliphile ATP synthase at high pH depends upon specific adaptations of both the respiratory chain complexes and the ATP synthase (5–9). Adaptations of the ATP synthase to function at alkaline pH were found in the membrane-embedded F o complex (5, 8–11). In the c- subunit of alkaliphiles, which forms the c 13 rotor ring, two specific motifs were described.…”