Membrane Na+-pyrophosphatases can transport protons at low sodium concentrations.

Membrane-bound Na(+)-pyrophosphatase (Na(+)-PPase), working in parallel with the corresponding ATP-energized pumps, catalyzes active Na(+) transport in bacteria and archaea. Each ~75-kDa subunit of homodimeric Na(+)-PPase forms an unusual funnel-like structure with a catalytic site in the cytoplasmic part and a hydrophilic gated channel in the membrane. Here, we show that at subphysiological Na(+) concentrations (<5 mM), the Na(+)-PPases of Chlorobium limicola, four other bacteria, and one archaeon additionally exhibit an H(+)-pumping activity in inverted membrane vesicles prepared from recombinant Escherichia coli strains. H(+) accumulation in vesicles was measured with fluorescent pH indicators. At pH 6.2-8.2, H(+) transport activity was high at 0.1 mM Na(+) but decreased progressively with increasing Na(+) concentrations until virtually disappearing at 5 mM Na(+). In contrast, (22)Na(+) transport activity changed little over a Na(+) concentration range of 0.05-10 mM. Conservative substitutions of gate Glu(242) and nearby Ser(243) and Asn(677) residues reduced the catalytic and transport functions of the enzyme but did not affect the Na(+) dependence of H(+) transport, whereas a Lys(681) substitution abolished H(+) (but not Na(+)) transport. All four substitutions markedly decreased PPase affinity for the activating Na(+) ion. These results are interpreted in terms of a model that assumes the presence of two Na(+)-binding sites in the channel: one associated with the gate and controlling all enzyme activities and the other located at a distance and controlling only H(+) transport activity. The inherent H(+) transport activity of Na(+)-PPase provides a rationale for its easy evolution toward specific H(+) transport.