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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 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.

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.

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.

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%.