Inhibitors of a Na⁺-pumping NADH-ubiquinone oxidoreductase play multiple roles to block enzyme function

The Na⁺-pumping NADH-ubiquinone (UQ) oxidoreductase (Na⁺-NQR) is present in the respiratory chain of many pathogenic bacteria and is thought to be a promising antibiotic target. Whereas many details of Na⁺-NQR structure and function are known, the mechanisms of action of potent inhibitors is not well-understood; elucidating the mechanisms would not only advance drug design strategies but might also provide insights on a terminal electron transfer from riboflavin to UQ. To this end, we performed photoaffinity labeling experiments using photoreactive derivatives of two known inhibitors, aurachin and korormicin, on isolated Vibrio cholerae Na⁺-NQR. The inhibitors labeled the cytoplasmic surface domain of the NqrB subunit including a protruding N-terminal stretch, which may be critical to regulate the UQ reaction in the adjacent NqrA subunit. The labeling was blocked by short-chain UQs such as ubiquinone-2. The photolabile group (2-aryl-5-carboxytetrazole (ACT)) of these inhibitors reacts with nucleophilic amino acids, so we tested mutations of nucleophilic residues in the labeled region of NqrB, such as Asp49 and Asp52 (to Ala), and observed moderate decreases in labeling yields, suggesting that these residues are involved in the interaction with ACT. We conclude that the inhibitors interfere with the UQ reaction in two ways: the first is blocking structural rearrangements at the cytoplasmic interface between NqrA and NqrB, and the second is the direct obstruction of UQ binding at this interfacial area. Unusual competitive behavior between the photoreactive inhibitors and various competitors corroborates our previous proposition that there may be two inhibitor binding sites in Na⁺-NQR

Membrane Topology Mapping of the Na(+)-Pumping NADH: Quinone Oxidoreductase from Vibrio cholerae by PhoA- Green Fluorescent Protein Fusion Analysis

The membrane topologies of the six subunits of Na(+)-translocating NADH:quinone oxidoreductase (Na(+)-NQR) from Vibrio cholerae were determined by a combination of topology prediction algorithms and the construction of C-terminal fusions. Fusion expression vectors contained either bacterial alkaline phosphatase (phoA) or green fluorescent protein (gfp) genes as reporters of periplasmic and cytoplasmic localization, respectively. A majority of the topology prediction algorithms did not predict any transmembrane helices for NqrA. A lack of PhoA activity when fused to the C terminus of NqrA and the observed fluorescence of the green fluorescent protein C-terminal fusion confirm that this subunit is localized to the cytoplasmic side of the membrane. Analysis of four PhoA fusions for NqrB indicates that this subunit has nine transmembrane helices and that residue T236, the binding site for flavin mononucleotide (FMN), resides in the cytoplasm. Three fusions confirm that the topology of NqrC consists of two transmembrane helices with the FMN binding site at residue T225 on the cytoplasmic side. Fusion analysis of NqrD and NqrE showed almost mirror image topologies, each consisting of six transmembrane helices; the results for NqrD and NqrE are consistent with the topologies of Escherichia coli homologs YdgQ and YdgL, respectively. The NADH, flavin adenine dinucleotide, and Fe-S center binding sites of NqrF were localized to the cytoplasm. The determination of the topologies of the subunits of Na(+)-NQR provides valuable insights into the location of cofactors and identifies targets for mutagenesis to characterize this enzyme in more detail. The finding that all the redox cofactors are localized to the cytoplasmic side of the membrane is discussed

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Membrane topology mapping of the Na +-pumping NADH:quinone oxidoreductase from Vibrio cholerae by PhoA/GFP fusion analysi

The Electron Transfer Pathway of the Na+-pumping NADH:Quinone Oxidoreductase from Vibrio cholerae*

The Na+-pumping NADH:quinone oxidoreductase (Na+-NQR) is the only respiratory enzyme that operates as a Na+ pump. This redox-driven Na+ pump is amenable to experimental approaches not available for H+ pumps, providing an excellent system for mechanistic studies of ion translocation. An understanding of the internal electron transfer steps and their Na+ dependence is an essential prerequisite for such studies. To this end, we analyzed the reduction kinetics of the wild type Na+-NQR, as well as site-directed mutants of the enzyme, which lack specific cofactors. NADH and ubiquinol were used as reductants in separate experiments, and a full spectrum UV-visible stopped flow kinetic method was employed. The results make it possible to define the complete sequence of redox carriers in the electrons transfer pathway through the enzyme. Electrons flow from NADH to quinone through the FAD in subunit F, the 2Fe-2S center, the FMN in subunit C, the FMN in subunit B, and finally riboflavin. The reduction of the FMNC to its anionic flavosemiquinone state is the first Na+-dependent process, suggesting that reduction of this site is linked to Na+ uptake. During the reduction reaction, two FMNs are transformed to their anionic flavosemiquinone in a single kinetic step. Subsequently, FMNC is converted to the flavohydroquinone, accounting for the single anionic flavosemiquinone radical in the fully reduced enzyme. A model of the electron transfer steps in the catalytic cycle of Na+-NQR is presented to account for the kinetic and spectroscopic data

Multiple horizontal gene transfer (HGT) events involved in the dispersal of the <i>nqr</i> operon.

<p>Our data indicate that Na⁺-NQR appeared in the common ancestor of the Chlorobi and Bacteroidetes groups. The operon was transferred by HGT to Chlamydiae and Planctomyces, and to the ancestor of α, β, γ and δ–proteobacteria, after this lineage split from ε -proteobacteria. The evidence indicates that both Desulfobacteraceae (δ–proteobacteria) and the ancestor of Burkholderales, Hydrogenophilales and Neisseriales (β–proteobacteria), lost the <i>nqr</i> operon and was reacquired later. In the case of Desulfobacterales, from Chlorobi, and in the case of Neisseriales, from pathogenic Pasteurelalles (γ–proteobacteria).</p

Origin and Evolution of the Sodium -Pumping NADH: Ubiquinone Oxidoreductase

<div><p>The sodium -pumping NADH: ubiquinone oxidoreductase (Na⁺-NQR) is the main ion pump and the primary entry site for electrons into the respiratory chain of many different types of pathogenic bacteria. This enzymatic complex creates a transmembrane gradient of sodium that is used by the cell to sustain ionic homeostasis, nutrient transport, ATP synthesis, flagellum rotation and other essential processes. Comparative genomics data demonstrate that the <i>nqr</i> operon, which encodes all Na⁺-NQR subunits, is found in a large variety of bacterial lineages with different habitats and metabolic strategies. Here we studied the distribution, origin and evolution of this enzymatic complex. The molecular phylogenetic analyses and the organizations of the <i>nqr</i> operon indicate that Na⁺-NQR evolved within the Chlorobi/Bacteroidetes group, after the duplication and subsequent neofunctionalization of the operon that encodes the homolog RNF complex. Subsequently, the <i>nqr</i> operon dispersed through multiple horizontal transfer events to other bacterial lineages such as Chlamydiae, Planctomyces and α, β, γ and δ -proteobacteria. Considering the biochemical properties of the Na⁺-NQR complex and its physiological role in different bacteria, we propose a detailed scenario to explain the molecular mechanisms that gave rise to its novel redox- dependent sodium -pumping activity. Our model postulates that the evolution of the Na⁺-NQR complex involved a functional divergence from its RNF homolog, following the duplication of the <i>rnf</i> operon, the loss of the <i>rnfB</i> gene and the recruitment of the reductase subunit of an aromatic monooxygenase.</p></div