Localization of ubiquinone-8 in the Na+-pumping NADH:quinone oxidoreductase from Vibrio cholerae

Na(+) is the second major coupling ion at membranes after protons, and many pathogenic bacteria use the sodium-motive force to their advantage. A prominent example is Vibrio cholerae, which relies on the Na(+)-pumping NADH:quinone oxidoreductase (Na(+)-NQR) as the first complex in its respiratory chain. The Na(+)-NQR is a multisubunit, membrane-embedded NADH dehydrogenase that oxidizes NADH and reduces quinone to quinol. Existing models describing redox-driven Na(+) translocation by the Na(+)-NQR are based on the assumption that the pump contains four flavins and one FeS cluster. Here we show that the large, peripheral NqrA subunit of the Na(+)-NQR binds one molecule of ubiquinone-8. Investigations of the dynamic interaction of NqrA with quinones by surface plasmon resonance and saturation transfer difference NMR reveal a high affinity, which is determined by the methoxy groups at the C-2 and C-3 positions of the quinone headgroup. Using photoactivatable quinone derivatives, it is demonstrated that ubiquinone-8 bound to NqrA occupies a functional site. A novel scheme of electron transfer in Na(+)-NQR is proposed that is initiated by NADH oxidation on subunit NqrF and leads to quinol formation on subunit NqrA.     

Na(+) translocation by bacterial NADH:quinone oxidoreductases: an extension to the complex-I family of primary redox pumps

The current knowledge on the Na(+)-translocating NADH:ubiquinone oxidoreductase of the Na(+)-NQR type from Vibrio alginolyticus, and on Na(+) transport by the electrogenic NADH:Q oxidoreductases from Escherichia coli and Klebsiella pneumoniae (complex I, or NDH-I) is summarized. A general mode of redox-linked Na(+) transport by NADH:Q oxidoreductases is proposed that is based on the electrostatic attraction of a positively charged Na(+) towards a negatively charged, enzyme-bound ubisemiquinone anion in a medium of low dielectricity. A structural model of the [2Fe-2S]- and FAD-carrying NqrF subunit of the Na(+)-NQR from V. alginolyticus based on ferredoxin and ferredoxin:NADP(+) oxidoreductase suggests that a direct participation of the Fe/S center in Na(+) transport is rather unlikely. A ubisemiquinone-dependent mechanism of Na(+) translocation is proposed that results in the transport of two Na(+) ions per two electrons transferred. Whereas this stoichiometry of the pump is in accordance with in vivo determinations of Na(+) transport by the respiratory chain of V. alginolyticus, higher (Na(+) or H(+)) transport stoichiometries are expected for complex I, suggesting the presence of a second coupling site.     

Sequencing and preliminary characterization of the Na+-translocating NADH:ubiquinone oxidoreductase from Vibrio harveyi

The Na(+)-translocating NADH: ubiquinone oxidoreductase (Na(+)-NQR) generates an electrochemical Na(+) potential driven by aerobic respiration. Previous studies on the enzyme from Vibrio alginolyticus have shown that the Na(+)-NQR has six subunits, and it is known to contain FAD and an FeS center as redox cofactors. In the current work, the enzyme from the marine bacterium Vibrio harveyi has been purified and characterized. In addition to FAD, a second flavin, tentatively identified as FMN, was discovered to be covalently attached to the NqrC subunit. The purified V. harveyi Na(+)-NQR was reconstituted into proteoliposomes. The generation of a transmembrane electric potential by the enzyme upon NADH:Q(1) oxidoreduction was strictly dependent on Na(+), resistant to the protonophore CCCP, and sensitive to the sodium ionophore ETH-157, showing that the enzyme operates as a primary electrogenic sodium pump. Interior alkalinization of the inside-out proteoliposomes due to the operation of the Na(+)-NQR was accelerated by CCCP, inhibited by valinomycin, and completely arrested by ETH-157. Hence, the protons required for ubiquinol formation must be taken up from the outside of the liposomes, which corresponds to the bacterial cytoplasm. The Na(+)-NQR operon from this bacterium was sequenced, and the sequence shows strong homology to the previously reported Na(+)-NQR operons from V. alginolyticus and Haemophilus influenzae. Homology studies show that a number of other bacteria, including a number of pathogenic species, also have an Na(+)-NQR operon.     

Recent progress in the Na(+)-translocating NADH-quinone reductase from the marine Vibrio alginolyticus

The respiratory chain of Gram-negative marine and halophilic bacteria has a Na(+)-dependent NADH-quinone reductase that functions as a primary Na(+) pump. The Na(+)-translocating NADH-quinone reductase (NQR) from the marine Vibrio alginolyticus is composed of six structural genes (nqrA to nqrF). The NqrF subunit has non-covalently bound FAD. There are conflicting results on the existence of other flavin cofactors. Recent studies revealed that the NqrB and NqrC subunits have a covalently bound flavin, possibly FMN, which is attached to a specified threonine residue. A novel antibiotic, korormicin, was found to specifically inhibit the NQR complex. From the homology search of the nqr operon, it was found that the Na(+)-pumping NQR complex is widely distributed among Gram-negative pathogenic bacteria.     

FMN is covalently attached to a threonine residue in the NqrB and NqrC subunits of Na(+)-translocating NADH-quinone reductase from Vibrio alginolyticus

The Na(+)-translocating NADH-quinone reductase (NQR) from Vibrio alginolyticus is composed of six subunits (NqrA to NqrF). We previously demonstrated that both NqrB and NqrC subunits contain a flavin cofactor covalently attached to a threonine residue. Fluorescent peptide fragments derived from the NqrB and NqrC subunits were applied to a matrix-assisted laser desorption ionization time-of-flight mass spectrometer, and covalently attached flavin was identified as FMN in both subunits. From post-source decay fragmentation analysis, it was concluded that FMN is attached by a phosphate group to Thr-235 in the NqrB subunit and to Thr-223 in the NqrC subunit. The phosphoester binding of FMN to a threonine residue reported here is a new type of flavin attachment to a polypeptide.     

Oxidant-induced formation of a neutral flavosemiquinone in the Na+-translocating NADH:Quinone oxidoreductase (Na+-NQR) from Vibrio cholerae

The Na(+)-translocating NADH:quinone oxidoreductase (Na(+)-NQR) from the human pathogen Vibrio cholerae is a respiratory flavo-FeS complex composed of the six subunits NqrA-F. The Na(+)-NQR was produced as His(6)-tagged protein by homologous expression in V. cholerae. The isolated complex contained near-stoichiometric amounts of non-covalently bound FAD (0.78 mol/mol Na(+)-NQR) and riboflavin (0.70 mol/mol Na(+)-NQR), catalyzed NADH-driven Na(+) transport (40 nmol Na(+)min(-1) mg(-1)), and was inhibited by 2-n-heptyl-4-hydroxyquinoline-N-oxide. EPR spectroscopy showed that Na(+)-NQR as isolated contained very low amounts of a neutral flavosemiquinone (10(-3) mol/mol Na(+)-NQR). Reduction with NADH resulted in the formation of an anionic flavosemiquinone (0.10 mol/mol Na(+)-NQR). Subsequent oxidation of the Na(+)-NQR with ubiquinone-1 or O(2) led to the formation of a neutral flavosemiquinone (0.24 mol/mol Na(+)-NQR). We propose that the Na(+)-NQR is fully oxidized in its resting state, and discuss putative schemes of NADH-triggered redox transitions.     

NMR Reveals Double Occupancy of Quinone-type Ligands in the Catalytic Quinone Binding Site of the Na+-translocating NADH:Quinone Oxidoreductase from Vibrio cholerae

The sodium ion-translocating NADH:quinone oxidoreductase (Na⁺-NQR) from the pathogen Vibrio cholerae exploits the free energy liberated during oxidation of NADH with ubiquinone to pump sodium ions across the cytoplasmic membrane. The Na⁺-NQR consists of four membrane-bound subunits NqrBCDE and the peripheral NqrF and NqrA subunits. NqrA binds ubiquinone-8 as well as quinones with shorter prenyl chains (ubiquinone-1 and ubiquinone-2). Here we show that the quinone derivative 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone (DBMIB), a known inhibitor of the bc₁ and b₆f complexes found in mitochondria and chloroplasts, also inhibits quinone reduction by the Na⁺-NQR in a mixed inhibition mode. Tryptophan fluorescence quenching and saturation transfer difference NMR experiments in the presence of Na⁺-NQR inhibitor (DBMIB or 2-n-heptyl-4-hydroxyquinoline N-oxide) indicate that two quinone analog ligands are bound simultaneously by the NqrA subunit with very similar interaction constants as observed with the holoenzyme complex. We conclude that the catalytic site of quinone reduction is located on NqrA. The two ligands bind to an extended binding pocket in direct vicinity to each other as demonstrated by interligand Overhauser effects between ubiquinone-1 and DBMIB or 2-n-heptyl-4-hydroxyquinoline N-oxide, respectively. We propose that a similar spatially close arrangement of the native quinone substrates is also operational in vivo, enhancing the catalytic efficiency during the final electron transfer steps in the Na⁺-NQR.     

Mutagenesis study of the 2Fe-2S center and the FAD binding site of the Na(+)-translocating NADH:ubiquinone oxidoreductase from Vibrio cholerae

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-1, 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.     

Purification and characterization of the recombinant Na(+)-translocating NADH:quinone oxidoreductase from Vibrio cholerae

The nqr operon from Vibrio cholerae, encoding the entire six-subunit, membrane-associated, Na(+)-translocating NADH:quinone oxidoreductase (Na(+)-NQR), was cloned under the regulation of the P(BAD) promoter. The enzyme was successfully expressed in V. cholerae. To facilitate molecular genetics studies of this sodium-pumping enzyme, a host strain of V. cholerae was constructed in which the genomic copy of the nqr operon was deleted. By using a vector containing a six-histidine tag on the carboxy terminus of the NqrF subunit, the last subunit in the operon, the recombinant enzyme was readily purified by affinity chromatography in a highly active form from detergent-solubilized membranes of V. cholerae. The recombinant enzyme has a high specific activity in the presence of sodium. NADH consumption was assessed at a turnover number of 720 electrons per second. When purified using dodecyl maltoside (DM), the isolated enzyme contains approximately one bound ubiquinone, whereas if the detergent LDAO is used instead, the quinone content of the isolated enzyme is negligible. Furthermore, the recombinant enzyme, purified with DM, has a relatively low rate of reaction with O(2) (10-20 s(-1)). In steady state turnover, the isolated, recombinant enzyme exhibits up to 5-fold stimulation by sodium and functions as a primary sodium pump, as reported previously for Na(+)()-NQR from other bacterial sources. When reconstituted into liposomes, the recombinant Na(+)-NQR generates a sodium gradient and a Delta Psi across the membrane. SDS-PAGE resolves all six subunits, two of which, NqrB and NqrC, contain covalently bound flavin. A redox titration of the enzyme, monitored by UV-visible spectroscopy, reveals three n = 2 redox centers and one n = 1 redox center, for which the presence of three flavins and a 2Fe-2S center can account. The V. cholerae Na(+)-NQR is well-suited for structural studies and for the use of molecular genetics techniques in addressing the mechanism by which NADH oxidation is coupled to the pumping of Na(+) across the membrane.     

Inactivation of the Na+-translocating NADH:ubiquinone oxidoreductase from Vibrio alginolyticus by reactive oxygen species.

The Na+-translocating NADH:quinone oxidoreductase (Na+-NQR) from Vibrio alginolyticus was inactivated by reactive oxygen species. Highest Na+-NQR activity was observed in anaerobically prepared membranes that exhibited 1:1 coupling of NADH oxidation and Q reduction activities (1.6 U x mg(-1)). Optical and EPR spectroscopy documented the presence of b-type cytochromes, a [2Fe-2S] cluster and an organic radical signal in anaerobically prepared membranes from V. alginolyticus. It is shown that the [2Fe-2S] cluster previously assigned to the Na+-NQR originates from the succinate dehydrogenase or the related enzyme fumarate reductase.     

Detection of the Na(+)-translocating NADH-quinone reductase in marine bacteria using a PCR technique

To examine the distribution of the Na(+)-translocating NADH-quinone reductase (Na(+)-NQR) among marine bacteria, we developed a simple screening method for the detection of this enzyme. By reference to the homologous sequences of the Na(+)-NQR operons from Vibrio alginolyticus and Haemophilus influenzae, a pair of primers was designed for amplification of a part of the sixth ORF (nqr6) of the Na(+)-NQR operon. When PCR was performed using genomic DNA from 13 marine bacteria, a 0.9-kbp fragment corresponding to nqr6 was amplified in 10 strains. Although there were three PCR-negative strains phylogenetically, based on the sequence of the 16S rRNA, these were placed far from the PCR-positive strains. No product was observed in the case of nonmarine bacteria. The nucleotide and predicted amino acid sequences of nqr6 were highly conserved among the PCR-positive marine bacteria. A phylogenetic analysis of marine bacteria, based on nqr6 sequencing, was performed.     

Roles of charged residues in the C-terminal region of PomA, a stator component of the Na+-driven flagellar motor

Bacterial flagellar motors use specific ion gradients to drive their rotation. It has been suggested that the electrostatic interactions between charged residues of the stator and rotor proteins are important for rotation in Escherichia coli. Mutational studies have indicated that the Na(+)-driven motor of Vibrio alginolyticus may incorporate interactions similar to those of the E. coli motor, but the other electrostatic interactions between the rotor and stator proteins may occur in the Na(+)-driven motor. Thus, we investigated the C-terminal charged residues of the stator protein, PomA, in the Na(+)-driven motor. Three of eight charge-reversing mutations, PomA(K203E), PomA(R215E), and PomA(D220K), did not confer motility either with the motor of V. alginolyticus or with the Na(+)-driven chimeric motor of E. coli. Overproduction of the R215E and D220K mutant proteins but not overproduction of the K203E mutant protein impaired the motility of wild-type V. alginolyticus. The R207E mutant conferred motility with the motor of V. alginolyticus but not with the chimeric motor of E. coli. The motility with the E211K and R232E mutants was similar to that with wild-type PomA in V. alginolyticus but was greatly reduced in E. coli. Suppressor analysis suggested that R215 may participate in PomA-PomA interactions or PomA intramolecular interactions to form the stator complex.     

Quinone reduction by the Na+-translocating NADH dehydrogenase promotes extracellular superoxide production in Vibrio cholerae

The pathogenicity of Vibrio cholerae is influenced by sodium ions which are actively extruded from the cell by the Na(+)-translocating NADH:quinone oxidoreductase (Na(+)-NQR). To study the function of the Na(+)-NQR in the respiratory chain of V. cholerae, we examined the formation of organic radicals and superoxide in a wild-type strain and a mutant strain lacking the Na(+)-NQR. Upon reduction with NADH, an organic radical was detected in native membranes by electron paramagnetic resonance spectroscopy which was assigned to ubisemiquinones generated by the Na(+)-NQR. The radical concentration increased from 0.2 mM at 0.08 mM Na(+) to 0.4 mM at 14.7 mM Na(+), indicating that the concentration of the coupling cation influences the redox state of the quinone pool in V. cholerae membranes. During respiration, V. cholerae cells produced extracellular superoxide with a specific activity of 10.2 nmol min(-1) mg(-1) in the wild type compared to 3.1 nmol min(-1) mg(-1) in the NQR deletion strain. Raising the Na(+) concentration from 0.1 to 5 mM increased the rate of superoxide formation in the wild-type V. cholerae strain by at least 70%. Rates of respiratory H(2)O(2) formation by wild-type V. cholerae cells (30.9 nmol min(-1) mg(-1)) were threefold higher than rates observed with the mutant strain lacking the Na(+)-NQR (9.7 nmol min(-1) mg(-1)). Our study shows that environmental Na(+) could stimulate ubisemiquinone formation by the Na(+)-NQR and hereby enhance the production of reactive oxygen species formed during the autoxidation of reduced quinones.     

Reduced redox-dependent mechanism and glucose-mediated reversal in gentamicin-resistant Vibrio alginolyticus

Strategy of managing antibiotic-resistant Vibrio alginolyticus, a bacterial pathogen that threatens human health and animal farming, is not available due to the lack of knowledge about the underlying mechanism of antibiotic resistance. Here, we showed that gentamicin-resistant V. alginolyticus (VA-R_GEN) has four mutations on metabolism and one mutation on a two-component system by whole-genome and PCR-based sequencing, indicating the metabolic shift in VA-R_GEN. Thus, metabolic profile was investigated by GC–MS based metabolomics. Glucose was identified as a crucial biomarker, whose abundance was decreased in VA-R_GEN. Further analysis with iPath, and gene expression and enzyme activity of the pyruvate cycle (the P cycle) demonstrated a global depressed metabolic pathway network in VA-R_GEN. Consistently, NADH, sodium-pumping NADH:ubiquinone oxidoreductase (Na(+)-NQR) system, membrane potential and intracellular gentamicin were decreased in VA-R_GEN. These findings indicate that the reduced redox state contributes to antibiotic resistance. Interestingly, exogenous glucose potentiated gentamicin to efficiently kill VA-R_GEN through the promotion of the P cycle, NADH, membrane potential and intracellular gentamicin. The potentiation was further confirmed in a zebrafish model. These results indicate that the gentamicin resistance reduces the P cycle and Na(+)-NQR system and thereby decreases redox state, membrane potential and gentamicin uptake, which can be reversed by exogenous glucose.     

Differentially Expressed Outer Membrane Proteins of Vibrio alginolyticus in Response to Six Types of Antibiotics

Vibrio alginolyticus is an opportunistic pathogen that occasionally causes life-threatening infections in individuals and results in great losses in marine aquacultures of crustaceans and fish. Recently, antibiotic-resistant strains of the bacterium from clinical and environmental sources have been reported with increasing frequency. However, few reports were involved in the antibiotic resistance of this bacterium at molecular levels. In the present study, Western blotting was utilized to investigate altered OM proteins of V. alginolyticus in response to six types of antibiotics: erythromycin, kanamycin, tetracycline, streptomycin, nalidixic acid, and chloromycetin. Seventeen OM proteins have been reported here for the first time to be related to antibiotic resistance. They were porins OmpU, OmpN, putative OmpU and LamB; transport proteins VA0802, VA2212 (FadL) and VPA0860; TolC family TolC and VA1631; lipoprotein VA0449; OmpA family VPA1186 and VA0764; iron-regulated proteins OmpV, VPA1435, and VA2602; and receptor protein OmpK; hypothetical protein VA1475. Importantly, VA2212 was up-regulated in response to the five antibiotics except nalidixic acid, and VPA1186 was down-regulated in response to the six antibiotics in antibiotic-stressed bacteria. They might be potentially universal targets for designing the new drugs that inhibit multi-resistant bacteria. These findings suggested that parallel investigations into a bacterium responding to several types of antibiotics would be helpful not only for the further understanding of antibiotic-resistant mechanisms but also for the screening of valuable targets of new drugs controlling antibiotic-resistant bacteria.     

Insights into the mechanism of electron transfer and sodium translocation of the Na(+)-pumping NADH:quinone oxidoreductase

Na(+)-NQR is a unique energy-transducing complex, widely distributed among marine and pathogenic bacteria. It converts the energy from the oxidation of NADH and the reduction of quinone into an electrochemical Na(+)-gradient that can provide energy for the cell. Na(+)-NQR is not homologous to any other respiratory protein but is closely related to the RNF complex. In this review we propose that sodium pumping in Na(+)-NQR is coupled to the redox reactions by a novel mechanism, which operates at multiple sites, is indirect and mediated by conformational changes of the protein. This article is part of a Special Issue entitled: 17th European Bioenergetics Conference (EBEC 2012).     

The single NqrB and NqrC subunits in the Na(+)-translocating NADH: Quinone oxidoreductase (Na(+)-NQR) from Vibrio cholerae each carry one covalently attached FMN

The Na(+)-translocating NADH:quinone oxidoreductase (Na(+)-NQR) is the prototype of a novel class of flavoproteins carrying a riboflavin phosphate bound to serine or threonine by a phosphodiester bond to the ribityl side chain. This membrane-bound, respiratory complex also contains one non-covalently bound FAD, one non-covalently bound riboflavin, ubiquinone-8 and a [2Fe-2S] cluster. Here, we report the quantitative analysis of the full set of flavin cofactors in the Na(+)-NQR and characterize the mode of linkage of the riboflavin phosphate to the membrane-bound NqrB and NqrC subunits. Release of the flavin by β-elimination and analysis of the cofactor demonstrates that the phosphate group is attached at the 5'-position of the ribityl as in authentic FMN and that the Na(+)-NQR contains approximately 1.7mol covalently bound FMN per mol non-covalently bound FAD. Therefore, each of the single NqrB and NqrC subunits in the Na(+)-NQR carries a single FMN. Elimination of the phosphodiester bond yields a dehydro-2-aminobutyrate residue, which is modified with β-mercaptoethanol by Michael addition. Proteolytic digestion followed by mass determination of peptide fragments reveals exclusive modification of threonine residues, which carry FMN in the native enzyme. The described reactions allow quantification and localization of the covalently attached FMNs in the Na(+)-NQR and in related proteins belonging to the Rhodobacter nitrogen fixation (RNF) family of enzymes. This article is part of a Special Issue entitled: 17th European Bioenergetics Conference (EBEC 2012).     

Korormicin insensitivity in Vibrio alginolyticus is correlated with a single point mutation of Gly-140 in the NqrB subunit of the Na(+)-translocating NADH-quinone reductase

Na(+)-translocating NADH-quinone reductase (NQR) from the marine Vibrio alginolyticus is strongly inhibited by a new antibiotic korormicin. Korormicin specifically inhibits the Na(+)-dependent reaction of the NQR complex and acts as a purely non-competitive inhibitor for Q-1 with the inhibitor constant of 82 pM. Korormicin-resistant mutants were isolated from V. alginolyticus and the NQR complex was purified from a mutant KR2. Similar to 2-n-heptyl-4-hydroxyquinoline N-oxide (HQNO), korormicin acted as a purely noncompetitive inhibitor to the NQR complex from the mutant KR2, but the inhibitor constant increased to 8 microM, which is 10(5)-fold higher than that of the wild-type NQR complex. The inhibitor constant of HQNO, however, was only slightly affected by the acquisition of korormicin resistance. The spontaneous mutation was caused by a single mutation of G-422 to T-422 in the nucleotide sequence of the nqrB gene, which resulted in the conversion of Gly-140 to Val-140. Thus, Gly-140 seems to play an important role for the binding of korormicin to the NqrB subunit. The fact that korormicin is a purely noncompetitive inhibitor for Q-1 strongly supports the presence of one of Q-1 binding sites in the NqrB subunit, which also has a covalently bound FMN at Thr-235.     

NADH oxidation by the Na+-translocating NADH:quinone oxidoreductase from Vibrio cholerae: functional role of the NqrF subunit

The Na(+)-translocating NADH:quinone oxidoreductase from Vibrio cholerae is a six subunit enzyme containing four flavins and a single motif for the binding of a Fe-S cluster on its NqrF subunit. This study reports the production of a soluble variant of NqrF (NqrF') and its individual flavin and Fe-S-carrying domains using V. cholerae or Escherichia coli as expression hosts. NqrF' and the flavin domain each contain 1 mol of FAD/mol of enzyme and exhibit high NADH oxidation activity (20,000 micromol min(-1) mg(-1)). EPR, visible absorption, and circular dichroism spectroscopy indicate that the Fe-S cluster in NqrF' and its Fe-S domain is related to 2Fe ferredoxins of the vertebrate-type. The addition of NADH to NqrF' results in the formation of a neutral flavosemiquinone and a partial reduction of the Fe-S cluster. The NqrF subunit harbors the active site of NADH oxidation and acts as a converter between the hydride donor NADH and subsequent one-electron reaction steps in the Na(+)-translocating NADH:quinone oxidoreductase complex. The observed electron transfer NADH --> FAD --> [2Fe-2S] in NqrF requires positioning of the FAD and the Fe-S cluster in close proximity in accordance with a structural model of the subunit.     

Identification and antibody-therapeutic targeting of chloramphenicol-resistant outer membrane proteins in Escherichia coli

Bacterial resistance to an antibiotic may result from survival in a suddenly strong antibiotic or in sub-minimum inhibitory concentration of the drug. Their shared proteins responsible for the resistance should be potential targets for designing new drugs to inhibit the growth of the antibiotic-resistant bacteria. In the current study, comparative proteomic methodologies were used for identification of sharedly altered outer membrane proteins (OM proteins) that are responsible for chloramphenical (CAP)-resistant Escherichia coli and for survival in medium with suddenly strong CAP treatment. Six differential OM proteins and another protein with unknown location were determined to be sharedly CAP-resistant-related proteins with the use of 2-DE/MS, Western blotting and gene mutant methods, in which TolC, OmpT, OmpC, and OmpW were critically altered proteins and potential targets for designing of the new drugs. Furthermore, a novel method of specific antibody combating bacterial growth was developed on these OM proteins. Only anti-TolC showed a very significant inhibition on bacterial growth in medium with CAP when antisera to TolC, OmpC, OmpT, and OmpW were separately utilized. The growth of CAP-resistant E. coli and its original strain was completely inhibited when they bound with anti-TolC and survived in 1/8 MIC of CAP. This observed result is basically the same to the finding that DeltatolC was survived in the same concentration of the antibiotic. Our study demonstrates that the enhancement of expression of antibody target with antibiotic could be very effective approach compared to using a drug alone, which highlights a potential way for treatment of infection by antibiotic-resistant bacteria.