Search

The Na+-pumping NADH-quinone oxidoreductase (Na+-NQR) is the first enzyme of the respiratory chain and the main ion transporter in many marine and pathogenic bacteria, including Vibrio cholerae The V. cholerae Na+-NQR has been extensively studied, but its binding sites for ubiquinone and inhibitors remain controversial. Here, using a photoreactive ubiquinone PUQ-3 as well as two aurachin-type inhibitors [125I]PAD-1 and [125I]PAD-2 and photoaffinity labeling experiments on the isolated enzyme, we demonstrate that the ubiquinone ring binds to the NqrA subunit in the regions Leu-32-Met-39 and Phe-131-Lys-138, encompassing the rear wall of a predicted ubiquinone-binding cavity. The quinolone ring and alkyl side chain of aurachin bound to the NqrB subunit in the regions Arg-43-Lys-54 and Trp-23-Gly-89, respectively. These results indicate that the binding sites for ubiquinone and aurachin-type inhibitors are in close proximity but do not overlap one another. Unexpectedly, although the inhibitory effects of PAD-1 and PAD-2 were almost completely abolished by certain mutations in NqrB (i.e. G140A and E144C), the binding reactivities of [125I]PAD-1 and [125I]PAD-2 to the mutated enzymes were unchanged compared with those of the wild-type enzyme. We also found that photoaffinity labeling by [125I]PAD-1 and [125I]PAD-2, rather than being competitively suppressed in the presence of other inhibitors, is enhanced under some experimental conditions. To explain these apparently paradoxical results, we propose models for the catalytic reaction of Na+-NQR and its interactions with inhibitors on the basis of the biochemical and biophysical results reported here and in previous work.

The Na+-pumping NADH-ubiquinone oxidoreductase (Na+-NQR) couples electron transfer from NADH to ubiquinone with Na+-pumping, generating an electrochemical Na+ gradient that is essential for energy-consuming reactions in bacteria. Since Na+-NQR is exclusively found in prokaryotes, it is a promising target for highly selective antibiotics. However, the molecular mechanism of inhibition is not well-understood for lack of the atomic structural information about an inhibitor-bound state. Here we present cryo-electron microscopy structures of Na+-NQR from Vibrio cholerae with or without a bound inhibitor at 2.5- to 3.1-Å resolution. The structures reveal the arrangement of all six redox cofactors including a herein identified 2Fe-2S cluster located between the NqrD and NqrE subunits. A large part of the hydrophilic NqrF is barely visible in the density map, suggesting a high degree of flexibility. This flexibility may be responsible to reducing the long distance between the 2Fe-2S centers in NqrF and NqrD/E. Two different types of specific inhibitors bind to the N-terminal region of NqrB, which is disordered in the absence of inhibitors. The present study provides a foundation for understanding the function of Na+-NQR and the binding manner of specific inhibitors. The Na+-pumping NADH-ubiquinone oxidoreductase (Na+-NQR) is a unique respiratory enzyme found in many pathogenic bacteria. Here, the authors present high-resolution cryo-EM structures of Na+-NQR from V. cholerae with or without a bound inhibitor. [ABSTRACT FROM AUTHOR]

The membrane topologies of the six subunits of [Na.sup.+]-transiocating NADH:quinone oxidoreductase ([Na.sup.+]-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.sup.+]-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.

A model of the structure of subunit I of a cbb(sub 3) oxidase that was constructed using homology modeling, quantum chemistry and molecular dynamics is discussed. The model shows that a tyrosine residue structurally analogous to the active-site tyrosine in other oxidases is present in the cbb(sub 3) oxidases but that the tyrosine originates from a different transmembrane helix within the protein.

The three conserved residues in the FAD binding motif are mutated to alanine or leucine, and each of the four conserved cysteines in the iron-sulfur center motif is mutated to alanine. The results clearly demonstrate that these residues are necessary for binding the FAD and 2Fe-2S cluster, as expected from the sequence motifs.

EPR and ENDOR data, which show conclusively that the radical signals present in the oxidized and reduced forms of Na(super +) -NQR are both flavin semiquinones is presented. Irrespective of whether the neutral and anionic radical signals in Na(super +)-NQR arise from one or two flavins, a flavin semiquinone could play a role in the translocation of sodium ions by the enzyme.

The recombinant sodium-translocating NADH;quinone oxidoreductase, encoded by the nqr operon from Vibrio cholerae, shows high turnover and exhibits high specific activity in the presence of sodium, up to 5-fold stimulation, enabling the enzyme to function as sodium pump. The enzyme is composed of six subunits, two of which, NqrB and NqrC contain flavin covalently bound.

Research has been conducted on the genes in the Escgerichia coli genome similar to the genes encoding eukaryotic Na (super)+ /H (super)+ exchangers. The role of these E. coli genes in bacterial physiology has been investigated and the results are discussed.

Research reveals that the sodium-dependent step within the Na(sup)+ -tranlocating NADH:ubiquinone oxidoreductase is localized between flavin adenine dinucleotide and flavin mono nucleotide. Data also point out that NADH reduction of the flavin is dependent on sodium concentration.

Results show that aspartate-75 (D75) and its mutant form D75E of the Escherichia coli cytochrome bo(sub)3 exhibit interconversion to protonated-deprotonated forms depending upon the oxido-reduction state of the cytochrome. Data also indicate that the reduced bound ubiquinone facilitates proton uptake by D75 and shares it with ubiquinol.

The properties of NADH:ubiquinone oxidoreductase from Vibrio alginolyticus are described. This enzyme is a primary electrogenic sodium pump.

The Q(sub)o site of mitochondrial ubihydroquinone:cytochrome C oxidoreductase can contain only an inhibitor or a quinol but not both. The exit tunnel forces the hydrophobic tails of compounds to occupy the same space. Therefore one would be displaced by the other.

A glutamate residue at position 89 in subunit II of cytochrome bo(sub)3 from E. coli may regulate proton transit through the K-channel. This enzyme is a heme-copper oxidase, and contains proton-conducting channels in subunit I.

Quinol oxidation by the [bc.sub.1] complex of Rhodobacter sphaeroides occurs from an enzyme-substrate complex formed between quinol bound at the [Q.sub.o] site and the iron-sulfur protein (ISP) docked at an interface on cytochrome b. From the structure of the stigmatellin-containing mitochondrial complex, we suggest that hydrogen bonds to the two quinol hydroxyl groups, from Glu-272 of cytochrome b and His-161 of the ISP, help to stabilize the enzyme-substrate complex and aid proton release. Reduction of the oxidized ISP involves H transfer from quinol. Release of the proton occurs when the acceptor chain reoxidizes the reduced ISP, after domain movement to an interface on cytochrome [c.sub.1]. Effects of mutations to the ISP that change the redox potential and/or the pK on the oxidized form support this mechanism. Structures for the complex in the presence of inhibitors show two different orientations of Glu-272. In stigmatellin-containing crystals, the side chain points into the site, to hydrogen bond with a ring hydroxyl, while His-161 hydrogen bonds to the carbonyl group. In the native structure, or crystals containing myxothiazol or [Beta]-methoxyacrylate-type inhibitors, the Glu-272 side chain is rotated to point out of the site, to the surface of an external aqueous channel. Effects of mutation at this residue suggest that this group is involved in ligation of stigmatellin and quinol, but not quinone, and that the carboxylate function is essential for rapid turnover. [H.sup.+] transfer from semiquinone to the carboxylate side chain and rotation to the position found in the myxothiazol structure provide a pathway for release of the second proton.

A unique complementation system for NQO8 of Paracoccus denitrificans is developed. It is aimed at isolating cells with active NDH-1 mutants and lacking NDH-2 as well as inactive NDH-1 mutants that express the alternative enzyme. Mutations were introduced into the NQO8 subunit of NDH-1, a bacterial homologue of ND1. Results demonstrate that mutations in specific amino acids of the NQO8 subunit of NDH-1 from P. denitrificans may decrease or abolish the specific ubiquinone reductase activity of the enzyme.

Site-directed mutagenesis of the highly conserved tryptophan-129 and arginine-14 residues in cytochrome b subunit of Rhodobacter sphaeroides bc1 complex reveals that the two residues are vital for the stability and structure of the bc1 complex. Mutations affect the quinone reductase site, cytochromes bH and bL and the photosynthetic development of R. sphaeroides. The functions of the two residues are in agreement with the positions of the residues close to the cytoplasmic sides of the putative transmembrane helices C and B.

A study of the enzymology of different oxidase species and their functions in a cell helps determine the functions of the oxidases in bacterial repiratory chains. Respiratory oxidases reduce 90% of molecular oxygen in the biosphere of the earth. Bacterial repiratory systems consist of several terminal oxidases and the single cytochrome a oxidase, which is characteristic of eukaryotic systems, is absent in these bacteria.

The area binding the 2Fe-2S group of the Rieske subunit of the ubiquinol:cytochrome C2 oxidoreductase of Rhodobacter sphaeroides, is found in site-directed mutations of conserved residues. Glycine is replaced with aspartate to analyze the thermodynamic reaction and the kinetics of ubiquinol:cytochrome C2 oxidoreductase on flash activation of the photosynthetic chain. The quinol oxidizing site shows the presence of the impaired catalyst in the mutant complex. The cytochrome subunit does not affect the basic structure of the 2Fe-2S group.

The effect of site-specific mutations on the cytochrome b subunit of the bc1 complex of Rhodobacter sphaeroides was investigated. Mutation was aimed at the residues which were in proximity to the quinone reductase site. REsults indicated that the mutants maintained a functional quinol oxidizing site although the rate of cytochrome bh reoxidation was variably affected. A critical defect was observed in the bc1 complex due to deficient reoxidation of cytochrome bh by ubiquinone. This in turn, impedes electron transport through the cytochromes.