title: Membrane Na+-pyrophosphatases can transport protons at low sodium concentrations.
abstract: 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.
authors.affiliation: From the Department of Biochemistry, University of Turku, FIN-20014 Turku, Finland and.
authors.forename: Heidi H
authors.initials: HH
authors.lastname: Luoto
authors.forename: Erika
authors.initials: E
authors.lastname: Nordbo
authors.forename: Alexander A
authors.initials: AA
authors.lastname: Baykov
authors.forename: Reijo
authors.initials: R
authors.lastname: Lahti
authors.forename: Anssi M
authors.initials: AM
authors.lastname: Malinen
mesh_terms: D019943:Amino Acid Substitution; D019843:Archaeal Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D025243:Bacterial Proton-Translocating ATPases / Q000378:metabolism; D001693:Biological Transport, Active; D041883:Chlorobium / Q000201:enzymology* / Q000235:genetics; D043564:Inorganic Pyrophosphatase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D007700:Kinetics; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D011487:Protein Conformation; D011522:Protons; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012964:Sodium / Q000378:metabolism
chemical_list: D019843:Archaeal Proteins; D001426:Bacterial Proteins; D011522:Protons; D011994:Recombinant Proteins; D012964:Sodium; D025243:Bacterial Proton-Translocating ATPases; D043564:Inorganic Pyrophosphatase
keywords: Bioenergetics; Enzyme Kinetics; Enzyme Mechanisms; Membrane Energetics; Membrane Proteins; Membrane Pyrophosphatase; Molecular Evolution; Proton Transport; Pyrophosphate; Sodium Transport