A mutation in Na(+)-NQR uncouples electron flow from Na(+) translocation in the presence of K(+).

The sodium-pumping NADH:ubiquinone oxidoreductase (Na(+)-NQR) is a bacterial respiratory enzyme that obtains energy from the redox reaction between NADH and ubiquinone and uses this energy to create an electrochemical Na(+) gradient across the cell membrane. A number of acidic residues in transmembrane helices have been shown to be important for Na(+) translocation. One of these, Asp-397 in the NqrB subunit, is a key residue for Na(+) uptake and binding. In this study, we show that when this residue is replaced with asparagine, the enzyme acquires a new sensitivity to K(+); in the mutant, K(+) both activates the redox reaction and uncouples it from the ion translocation reaction. In the wild-type enzyme, Na(+) (or Li(+)) accelerates turnover while K(+) alone does not activate. In the NqrB-D397N mutant, K(+) accelerates the same internal electron transfer step (2Fe-2S → FMNC) that is accelerated by Na(+). This is the same step that is inhibited in mutants in which Na(+) uptake is blocked. NqrB-D397N is able to translocate Na(+) and Li(+), but when K(+) is introduced, no ion translocation is observed, regardless of whether Na(+) or Li(+) is present. Thus, this mutant, when it turns over in the presence of K(+), is the first, and currently the only, example of an uncoupled Na(+)-NQR. The fact the redox reaction and ion pumping become decoupled from each other only in the presence of K(+) provides a switch that promises to be a useful experimental tool.

Dehalococcoides mccartyi strain JNA dechlorinates multiple chlorinated phenols including pentachlorophenol and harbors at least 19 reductive dehalogenase homologous genes.

Pentachlorophenol and other chlorinated phenols are highly toxic ubiquitous environmental pollutants. Using gas chromatographic analysis we determined that Dehalococcoides mccartyi strain JNA in pure culture dechlorinated pentachlorophenol to 3,5-dichlorophenol (DCP) via removal of the ortho and para chlorines in all of the three possible pathways. In addition, JNA dechlorinated 2,3,4,6-tetrachlorophenol via 2,4,6-trichlorophenol (TCP) and 2,4,5-TCP to 2,4-DCP and 3,4-DCP, respectively, and dechlorinated 2,3,6-TCP to 3-chlorophenol (CP) via 2,5-DCP. JNA converted 2,3,4-TCP to 3,4-DCP and 2,4-DCP by ortho and meta dechlorination, respectively. 2,3-DCP was dechlorinated to 3-CP, and, because cultures using it could be transferred with a low inoculum (0.5 to 1.5% vol/vol), it may act as an electron acceptor to support growth. Using PCR amplification with targeted and degenerate primers followed by cloning and sequencing, we determined that JNA harbors at least 19 reductive dehalogenase homologous (rdh) genes including orthologs of pcbA4 and pcbA5, pceA, and mbrA, but not tceA or vcrA. Many of these genes are shared with D. mccartyi strains CBDB1, DCMB5, GT, and CG5. Strain JNA has previously been shown to extensively dechlorinate the commercial polychlorinated biphenyl (PCB) mixture Aroclor 1260. Collectively the data suggest that strain JNA may be well adapted to survive in sites contaminated with chlorinated aromatics and may be useful for in situ bioremediation.

Aspartic acid 397 in subunit B of the Na+-pumping NADH:quinone oxidoreductase from Vibrio cholerae forms part of a sodium-binding site, is involved in cation selectivity, and affects cation-binding site cooperativity.

The Na(+)-pumping NADH:quinone complex is found in Vibrio cholerae and other marine and pathogenic bacteria. NADH:ubiquinone oxidoreductase oxidizes NADH and reduces ubiquinone, using the free energy released by this reaction to pump sodium ions across the cell membrane. In a previous report, a conserved aspartic acid residue in the NqrB subunit at position 397, located in the cytosolic face of this protein, was proposed to be involved in the capture of sodium. Here, we studied the role of this residue through the characterization of mutant enzymes in which this aspartic acid was substituted by other residues that change charge and size, such as arginine, serine, lysine, glutamic acid, and cysteine. Our results indicate that NqrB-Asp-397 forms part of one of the at least two sodium-binding sites and that both size and charge at this position are critical for the function of the enzyme. Moreover, we demonstrate that this residue is involved in cation selectivity, has a critical role in the communication between sodium-binding sites, by promoting cooperativity, and controls the electron transfer step involved in sodium uptake (2Fe-2S → FMNC).

The role and specificity of the catalytic and regulatory cation-binding sites of the Na+-pumping NADH:quinone oxidoreductase from Vibrio cholerae.

The Na(+)-translocating NADH:quinone oxidoreductase is the entry site for electrons into the respiratory chain and the main sodium pump in Vibrio cholerae and many other pathogenic bacteria. In this work, we have employed steady-state and transient kinetics, together with equilibrium binding measurements to define the number of cation-binding sites and characterize their roles in the enzyme. Our results show that sodium and lithium ions stimulate enzyme activity, and that Na(+)-NQR enables pumping of Li(+), as well as Na(+) across the membrane. We also confirm that the enzyme is not able to translocate other monovalent cations, such as potassium or rubidium. Although potassium is not used as a substrate, Na(+)-NQR contains a regulatory site for this ion, which acts as a nonessential activator, increasing the activity and affinity for sodium. Rubidium can bind to the same site as potassium, but instead of being activated, enzyme turnover is inhibited. Activity measurements in the presence of both sodium and lithium indicate that the enzyme contains at least two functional sodium-binding sites. We also show that the binding sites are not exclusively responsible for ion selectivity, and other steps downstream in the mechanism also play a role. Finally, equilibrium-binding measurements with (22)Na(+) show that, in both its oxidized and reduced states, Na(+)-NQR binds three sodium ions, and that the affinity for sodium is the same for both of these states.