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[Na.sup.+]-NQR is a unique respiratory enzyme that couples the free energy of electron transfer reactions to electrogenic pumping of sodium across the cell membrane. This enzyme is found in many marine and pathogenic bacteria where it plays an analogous role to the [H.sup.+] -pumping complex I. It has generally been assumed that the sodium pump of [Na.sup.+]-NQR operates on the basis of thermodynamic coupling between reduction of a single redox cofactor and the binding of sodium at a nearby site. In this study, we have defined the coupling to sodium translocation of individual steps in the redox reaction of [Na.sup.+]-NQR. Sodium uptake takes place in the reaction step in which an electron moves from the 2Fe-2S center to [FMN.sub.c] while the translocation of sodium across the membrane dielectric (and probably its release into the external medium) occurs when an electron moves from [FMN.sub.B] to riboflavin. This argues against a single-site coupling model because the redox steps that drive these two parts of the sodium pumping process do not have any redox cofactor in common. The significance of these results for the mechanism of coupling is discussed, and we proposed that [Na.sup.+]-NQR operates through a novel mechanism based on kinetic coupling, mediated by conformational changes. electron transfer | ion coupling | sodium transport doi/10.1073/pnas.1002866107

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.

Flavins are cofactors in many electron-transfer enzymes. Typically, two types of flavins perform this role: 5'-phosphoriboflavin (FMN) and flavin-adenine dinucleotide (FAD). Both of these are riboflavin derivatives, but riboflavin itself has never been reported to be an enzyme-bound component. We now report that tightly bound riboflavin is a component of the NADH-driven sodium pump from Vibrio cholerae.

The occurrence of kinetic stability of proteins is analyzed by using a diagonal two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis method. The results have shown that kinetic stability is prevalent in prokaryotes, but uncommon in eukaryotic organisms, thus indicating that this property is important for the adaptation and survival of less complex prokaryotic organisms.

The mutants of acid residues located in the transmembrane helices of subunits B, D, and E of the [Na.sup.+]-pumping NADH:quinone oxidoreductase ([Na.sup.+]-NQR) are analyzed to understand the negative charge of these residues which facilitated binding and translocation in sodium. The mutation results for various NQR residues are found to be consistent with the possible role in sodium conductance pathways.

The genes for RnfD and RnfG flavoproteins from Vibrio cholerae are cloned and expressed individually to understand the binding mechanism in threonine-175 bound to RnfG and threonine-187 bound to RnfD. The results obtained provide insight into the location of RnfG and RnfD in the periplasmic space and the mechanistic differences between the Rnf and [Na.sup.+]-NQR complexes.

Results reveal that surface-exposed glutamic acid E101(sub)II, in subunit II of cytochrome c oxidase from Rhodobacter sphaeroides, is the main entry point for protons going into the potassium-channel of the enzyme. Data indicate that any substitution for E1010(sub)II reduces cytochrome c oxidase activity by 8-29%.