A Mutation in Na⁺-NQR Uncouples Electron Flow from Na⁺ Translocation in the Presence of K⁺

Abstract

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.

The Kinetic Reaction Mechanism of the Vibrio cholerae Sodium-dependent NADH Dehydrogenase

“…These enzymes are multimeric complexes that are organized in modules that can act independently, allowing the operation of the ping-pong mechanism. This type of module organization is also found in Na ϩ -NQR, with the NADH dehydrogenase module located in the NqrF subunit (26,43), CoQ-1 reduction in the NqrB subunit (19,20), and sodium transport in the NqrB and NqrD/ NqrE subunits (14,15,17,30). The hexa-uni ping-pong mechanism is conformational in character, because the individual binding sites of the substrates are not preformed (or are inaccessible in certain states), which confirms our previous hypothesis that the enzyme undergoes multiple conformational changes (5,16,19,30).…”

“…The NqrB subunit contains the attachment site for one covalently bound FMN molecule (13), as well some of the structures involved in the transport of sodium, including one of the at least two sodium-binding sites (14 -17). Indeed, the mutation of aspartate residue 397 affects the positive cooperativity of the sites (15), changes the cation selectivity, and produces an uncoupling effect in the presence of potassium (17). Recent crystallographic data have shown that NqrB contains 10 transmembrane helices (12), with an organization that allows the transport of sodium.…”

<p>Metabolic mechanism of ceftazidime resistance in <em>Vibrio alginolyticus</em></p>

“…VA-R CAZ also show altered energy metabolism, deregulation of Na + -NQR, and low membrane potential, all factors that could potentiate and/or contribute to resistance to CAZ (note that Δ nqrA and Δ nqrF mutations are reported to decrease susceptibility to CAZ) 36. Consistent with this, membrane potential was reduced in VA-R CAZ , Δ nqrA , and Δ nqrF , and drug resistance of VA-S was potentiated in the presence of respiratory chain inhibitors.…”

Identification of the Catalytic Ubiquinone-binding Site of Vibrio cholerae Sodium-dependent NADH Dehydrogenase

“…Location of Na ϩ -NQR Catalytic UQ Binding Site, a Novel UQ Binding Motif-The catalytic mechanism of Na ϩ -NQR has been extensively studied, and the locations of most ligand and cofactor binding motifs have been identified, through site-directed mutagenesis, functional studies (5,8,16,17,20), and X-ray crystallography (12). Moreover, the electron transfer sequence and the segments of the catalytic cycle involved in sodium transfer have been thoroughly characterized (16,18,(21)(22)(23)(24)26).…”

“…Subunit B contains 10 transmembrane segments and one of the two covalently bound FMN cofactors (12)(13)(14). This subunit also plays an important role in sodium transport and carries one of the at least two sodium binding sites and an ion channel (15)(16)(17)(18). Subunit C has a large periplasmic domain that contains the second covalently bound FMN cofactor and a single transmembrane segment (12)(13)(14).…”