eTopic 14.1 ATP Synthesis at High pH

Alkaliphilic bacteria isolated from soda lakes, such as Lake Natron in Tanzania, grow at pH values as high as pH 14 while maintaining internal pH at 9–10. Thus, their pH is inverted, with 10,000-fold higher H+ concentration inside the cell. How do these alkaliphiles maintain a proton motive force driving protons inward? Maintaining internal pH several units lower than outside the cell seems incompatible with the generation of a proton potential that runs ATP synthase. So how do these organisms make ATP?

One model to explain ATP synthase coupling at high pH was proposed by Terry Krulwich, at Mount Sinai School of Medicine (Fig. 1). Krulwich proposed that protons pumped by the electron transport system are sequestered just outside the membrane and directed into the ATP synthase (Fig. 1B). This model (as yet unproven) would require a cell compartment, or trap, for protons outside the cell membrane or else some kind of direct connection between the ETS proton pumps and the ATP synthase.

The structure of the hypothetical proton coupling connection is unclear, but one prediction from the model is that the membrane-embedded Fo component of ATP synthase, which transduces proton flow, would have a form specialized for alkaliphilic conditions. Krulwich and colleagues compared the peptide sequences of Fo from an alkaliphilic species, Bacillus pseudofirmus OF4, with that of a neutralophilic species of the same genus, B. megaterium. Six amino acid differences were observed in the sequences of the membrane-embedded a and c subunits, which mediate the proton flux.

Mutant strains of the alkaliphile B. pseudofirmus were obtained, each of which had an amino acid substitution at one of sites 1–6 (Fig. 2). Four of the respective mutants failed to synthesize ATP when grown at high pH (mutation sites 1, 2, 4, and 6). This observation shows that the alkaliphile has evolved a form of ATP synthase specifically adapted to function at high external pH. It is consistent with a hypothesis that the alkaliphile’s cell membrane contains a special proton compartment or coupling device to drive ATP synthesis, although other explanations are possible.


Figure 1  Alkaliphiles maintain an inverted pH gradient.  At external pH several units higher than internal pH, how do alkaliphilic bacteria make ATP? A. Terry Krulwich, researcher on alkaliphilic bacteria. B. Proposed model for how protons pumped by the electron transport system might be sequestered just outside the membrane and directed into the ATP synthase. Figure courtesy of Terry A. Krulwich.


Figure 2  Mutations that inactivate ATP synthase in an alkaliphile.  A. ATP synthase structure in the alkaliphile B. pseudofirmus OF4, showing the membrane location of subunits a and c. B. Location of mutant residues in subunits a and c that inactivate the ATP synthase at high pH.