Membrane Na+-pyrophosphatases Can Transport Protons at Low Sodium Concentrations |
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Authors: | Heidi H Luoto Erika Nordbo Alexander A Baykov Reijo Lahti Anssi M Malinen |
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Institution: | From the ‡Department of Biochemistry, University of Turku, FIN-20014 Turku, Finland and ;the §Belozersky Institute of Physico-Chemical Biology, Lomonosov Moscow State University, Moscow 119899, Russia |
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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, 22Na+ transport activity changed little over a Na+ concentration range of 0.05–10 mm. Conservative substitutions of gate Glu242 and nearby Ser243 and Asn677 residues reduced the catalytic and transport functions of the enzyme but did not affect the Na+ dependence of H+ transport, whereas a Lys681 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. |
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Keywords: | Bioenergetics Enzyme Kinetics Enzyme Mechanisms Membrane Energetics Membrane Proteins Molecular Evolution Proton Transport Sodium Transport Membrane Pyrophosphatase Pyrophosphate |
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