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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 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.

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%.

Many marine and pathogenic bacteria have a unique sodium-translocating NADH:ubiquinone oxidoreductase (Na+-NQR), which generates an electrochemical Na+ gradient during aerobic respiration. Na+-NQR consists of six subunits (NqrA-F) and contains five known redox cofactors: two covalently bound FMNs, one noncovalently bound FAD, one riboflavin, and one 2Fe-2S center. A stable neutral flavin-semiquinone radical is observed in the air-oxidized enzyme, while the NADH- or dithionite-reduced enzyme exhibits a stable anionic flavin-semiquinone radical. The NqrF subunit has been implicated in binding of both the 2Fe-2S cluster and the FAD. Four conserved cysteines (C70, C76, C79, and C111) in NqrF match the canonical 2Fe-2S motif, and three conserved residues (R210, Y212, S246) have been predicted to be part of a flavin binding domain. In this work, these two motifs have been altered by site-directed mutagenesis of individual residues and are confirmed to be essential for binding, respectively, the 2Fe-2S cluster and FAD. EPR spectra of the FAD-deficient mutants in the oxidized and reduced forms exhibit neutral and anionic flavo-semiquinone radical signals, respectively, demonstrating that the FAD in NqrF is not the source of either radical signal. In both the FAD and 2Fe-2S center mutants the line widths of the neutral and anionic flavo-semiquinone EPR signals are unchanged from the wild-type enzyme, indicating that neither of these centers is nearby or coupled to the radicals. Measurements of steady-state turnover using NADH, Q-l, and the artificial electron acceptor ferricyanide strongly support an electron transport pathway model in which the noncovalently bound FAD in the NqrF subunit is the initial electron acceptor and electrons then flow to the 2Fe-2S center. [ABSTRACT FROM AUTHOR]

Examines the characterization of recombinant Na[sup +] in translocating NAD(P) H dehydrogenases from Vibrio cholerae. Regulation of the P[BAD] promoter; Creation of host strain for molecular genetics and delete nqy operon; Visualization of redox titration by ultraviolet ray in visible spectroscopy.

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. [ABSTRACT FROM AUTHOR]

The sodium-pumping NADH:quinone oxidoreductase (Na + -NQR) is a bacterial enzyme that oxidizes NADH, reduces ubiquinone, and translocates Na + across the membrane. We previously identified three acidic residues in the membrane-spanning helices, near the cytosol, NqrB-D397, NqrD-D133, and NqrE-E95, as candidates likely to be involved in Na + uptake, and replacement of any one of them by a non-acidic residue affects the Na + -dependent kinetics of the enzyme. Here, we have inquired further into the role of the NqrE-E95 residue by constructing a series of mutants in which this residue is replaced by amino acids with charges and/or sizes different from those of the glutamate of the wild-type enzyme. All of the mutants showed altered steady-state kinetics with the acceleration of turnover by Na + greatly diminished. Selected mutants were studied by other physical methods. Membrane potential measurements showed that NqrE-E95D and A are significantly less efficient in ion transport. NqrE-E95A, Q, and D were studied by transient kinetic measurements of the reduction of the enzyme by NADH. In all three cases, the results indicated inhibition of the electron-transfer step in which the FMN C becomes reduced. This is the first Na + -dependent step and is associated with Na + uptake by the enzyme. Electrochemical measurements on NqrE-E95Q showed that the Na + dependence of the redox potential of the FMN cofactors has been lost. The fact that the mutations at the NqrE-E95 site have specific effects related to translocation of Na + and Li + strongly indicates a definite role for NqrE-E95 in the cation transport process of Na + -NQR.

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.

In the respiratory chains of mitochondria and many aerobic prokaryotes, heme-copper oxidases are the terminal enzymes that couple the reduction of molecular oxygen to proton pumping, contributing to the protonmotive force. The cbb(3) oxidases belong to the superfamily of enzymes that includes all of the heme-copper oxidases. Sequence analysis indicates that the cbb(3) oxidases are missing an active-site tyrosine residue that is absolutely conserved in all other known heme-copper oxidases. In the other heme-copper oxidases, this tyrosine is known to be subject to an unusual post-translational modification and to play a critical role in the catalytic mechanism. The absence of this tyrosine in the cbb(3) oxidases raises the possibility that the cbb(3) oxidases utilize a different catalytic mechanism from that of the other members of the superfamily. Using homology modeling, quantum chemistry, and molecular dynamics, a model of the structure of subunit I of a cbb(3) oxidase (Vibrio cholerae) was constructed. The model predicts that a tyrosine residue structurally analogous to the active-site tyrosine in other oxidases is present in the cbb(3) oxidases but that the tyrosine originates from a different transmembrane helix within the protein. The predicted active-site tyrosine is conserved in the sequences of all of the known cbb(3) oxidases. Mutagenesis of the tyrosine to phenylalanine in the V. cholerae oxidase resulted in a fully assembled enzyme with nativelike structure but lacking catalytic activity. These findings strongly suggest that all of the heme-copper oxidases utilize the same catalytic mechanism and provide an unusual example in which a critical active-site residue originates from different places within the primary sequence for different members of the same superfamily.