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.

Figure 1

Scheme of the Na⁺-NQR complex showing the subunit composition and redox cofactors. NADH is oxidized by FAD in NqrF. Electrons move trough the enzyme to the final electron ubiquinone in the following order: FAD → 2Fe-2S center → FMN_C → FMN_B → riboflavin. The 2Fe-2S center → FMN_C step controls Na⁺ uptake, and the FMN_B → riboflavin step controls Na⁺ release. The arrows indicate the translocation of Na⁺ through NqrB, -D, and -E.

Figure 2

Dependence of the quinone reductase activity of NqrB-D397N on cation concentration for Na⁺ (●), Li⁺ (○), and K⁺ (■). The Co-Q1 red activity was in TEG buffer, containing 50 mM Tris-HCl (pH 8.0), 1 mM EDTA, 5% (v/v) glycerol, 0.05% (w/v) n-dodecyl β-d-maltoside, and NADH with different concentrations of NaCl, KCl, and LiCl. Saturating amounts of K₂-NADH and Co-Q1 were used (150 and 50 µM, respectively) Standard deviations are listed in Table 1.

Figure 3

Time course of reduction of the NqrB-D397N mutant by NADH (300 µM) in the presence of different concentrations of KCl at 575 nm (A) and 460 nm (B): no K⁺ (black), 25 mM KCl (blue), 50 mM KCl (cyan), 200 mM KCl (green), 300 mM KCl (orange), and 400 mM KCl (red). The inset shows the rate constant of the main K⁺-dependent phase at 575 nm as a function of K⁺ concentration. All concentrations after mixing.

Figure 4

Generation of membrane potential (ΔΨ) by NqrB-D397N reconstituted into proteolipsomes. The essay buffer contained 5 µM oxonol VI, 200 µM NADH, 100 µM CoQ-1, 300 mM sorbitol, 50 mM Tris-HCl (pH 7.5), and 1 mM EDTA, along with the cation(s) of interest (A) using 20 mM NaCl (black), 20 mM LiCl (red), and 20 mM KCl (blue) and (B) (top) NaCl titration (1 to 50 mM) and (bottom) effect of KCl (100 mM) on the membrane potential in the presence of 2 mM NaCl and 50 mM NaCl.

Figure 5

Scheme illustrating properties of the NqrB-D397N mutant compared to those of the wild-type enzyme. In red are shown the acidic residues involved in Na⁺ uptake. In the wild-type enzyme (top row, left to right), (i) electron flow is coupled to Na⁺ pumping, K⁺ binds to a different, allosteric site (bottom left corner) and accelerates the reaction (ii) in the absence of Na⁺, and electron flow is inhibited, confirming that the redox and pumping processes are coupled. In the NqrB-D397N mutant (bottom row, left to right), (iii) in the absence of K⁺, electron flow is still coupled to Na⁺ pumping; (iv) in the presence of K⁺ (in the absence of Na⁺), electron flow proceeds, but without ion pumping, meaning that coupling has been disrupted; and (v) K⁺ causes the mutant to run uncoupled, even in the presence of Na⁺. Of the acidic residues involved in cation uptake, shown in bold, only NqrB-D397 is associated with the modified binding site.