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PubMed2002

The vbs genes that direct synthesis of the siderophore vicibactin in Rhizobium leguminosarum: their expression in other genera requires ECF sigma factor RpoI.

A cluster of eight genes, vbsGSO, vbsADL, vbsC and vbsP, are involved in the synthesis of vicibactin, a cyclic, trihydroxamate siderophore made by the symbiotic bacterium Rhizobium leguminosarum. None of these vbs genes was required for symbiotic N2 fixation on peas or Vicia. Transcription of vbsC, vbsGSO and vbsADL (but not vbsP) was enhanced by growth in low levels of Fe. Transcription of vbsGSO and vbsADL, but not vbsP or vbsC, required the closely linked gene rpoI, which encodes an ECF sigma factor of RNA polymerase. Transfer of the cloned vbs genes, plus rpoI, to Rhodobacter, Paracoccus and Sinorhizobium conferred the ability to make vicibactin on these other genera. We present a biochemical genetic model of vicibactin synthesis, which accommodates the phenotypes of different vbs mutants and the homologies of the vbs gene products. In this model, VbsS, which is similar to many non-ribosomal peptide synthetase multienzymes, has a central role. It is proposed that VbsS activates L-N5-hydroxyornithine via covalent attachment as an acyl thioester to a peptidyl carrier protein domain. Subsequent VbsA-catalysed acylation of the hydroxyornithine, followed by VbsL-mediated epimerization and acetylation catalysed by VbsC, yields the vicibactin subunit, which is then trimerized and cyclized by the thioesterase domain of VbsS to give the completed siderophore.

Bacterial Proteins↗
PubMed2003

Electron transport to periplasmic nitrate reductase (NapA) of Wolinella succinogenes is independent of a NapC protein.

The rumen bacterium Wolinella succinogenes grows by respiratory nitrate ammonification with formate as electron donor. Whereas the enzymology and coupling mechanism of nitrite respiration is well known, nitrate reduction to nitrite has not yet been examined. We report here that intact cells and cell fractions catalyse nitrate and chlorate reduction by reduced viologen dyes with high specific activities. A gene cluster encoding components of a putative periplasmic nitrate reductase system (napA, G, H, B, F, L, D) was sequenced. The napA gene was inactivated by inserting a kanamycin resistance gene cassette. The resulting mutant did not grow by nitrate respiration and did not reduce nitrate during growth by fumarate respiration, in contrast to the wild type. An antigen was detected in wild-type cells using an antiserum raised against the periplasmic nitrate reductase (NapA) from Paracoccus pantotrophus. This antigen was absent in the W. succinogenes napA mutant. It is concluded that the periplasmic nitrate reductase NapA is the only respiratory nitrate reductase in W. succinogenes, although a second nitrate-reducing enzyme is apparently induced in the napA mutant. The nap cluster of W. succinogenes lacks a napC gene whose product is thought to function in quinol oxidation and electron transfer to NapA in other bacteria. The W. succinogenes genome encodes two members of the NapC/NirT family, NrfH and FccC. Characterization of corresponding deletion mutants indicates that neither of these two proteins is required for nitrate respiration. A mutant lacking the genes encoding respiratory nitrite reductase (nrfHA) had wild-type properties with respect to nitrate respiration. A model of the electron transport chain of nitrate respiration is proposed in which one or more of the napF, G, H and L gene products mediate electron transport from menaquinol to the periplasmic NapAB complex. Inspection of the W. succinogenes genome sequence suggests that ammonia formation from nitrate is catalysed exclusively by periplasmic respiratory enzymes.

Bacterial Proteins↗
PubMed1998

Occurrence, overexpression and partial purification of the protein (majastridin) corresponding to the URF6 gene of the Rhodobacter blasticus atp operon.

Antibodies were produced against two antigenic peptides of a protein, which was named majastridin, corresponding to the URF6 gene of the Rhodobacter blasticus atp operon [Tybulewicz, V. L. J., Falk, G. & Walker, J. E. (1984) J. Mol. Biol. 179, 185-214]. A protein band of the expected size is labelled by immunoblotting in Western blots containing the cytosolic fractions from Rb. blasticus and Paracoccus denitrificans but not from Escherichia coli or Rhodospirillum rubrum. Although the protein is present during the entire life cycle of a Rb. blasticus culture, it is most abundant early during the stationary phase. Plasmid constructs of the URF6 gene for overexpression in E. coli were made. These constructs were designed to obtain proteins both with and without His-tagging. In both cases, a protein product was visible in induced cells. The His-tagged protein was purified to 85% on a Ni column and, further, to at least 95% by anion-exchange chromatography. By N-terminal sequencing of the His-tagged protein, its identity was confirmed.

Antibodies, Bacterial↗
PubMed2001

Isolation and characterization of complex I, rotenone-sensitive NADH: ubiquinone oxidoreductase, from the procyclic forms of Trypanosoma brucei.

Additional characterization of complex I, rotenone-sensitive NADH:ubiquinone oxidoreductase, in the mitochondria of Trypanosoma brucei brucei has been obtained. Both proline:cytochrome c reductase and NADH:ubiquinone oxidoreductase of procyclic T. brucei were inhibited by the specific inhibitors of complex I rotenone, piericidin A, and capsaicin. These inhibitors had no effect on succinate: cytochrome c reductase activity. Antimycin A, a specific inhibitor of the cytochrome bc1 complex (ubiquinol:cytochrome c oxidoreductase), blocked almost completely cytochrome c reductase activity with either proline or succinate as electron donor, but had no inhibitory effect on NADH:ubiquinone oxidoreductase activity. The rotenone-sensitive NADH:ubiquinone oxidoreductase of procyclic T. brucei was partially purified by sucrose density centrifugation of mitochondria solubilized with dodecyl-beta-D-maltoside, with an approximately eightfold increase in specific activity compared to that of the mitochondrial membranes. Four polypeptides of the partially purified enzyme were identified as the homologous subunits of complex I (51 kDa, PSST, TYKY, and ND4) by immunoblotting with antibodies raised against subunits of Paracoccus denitrificans and against synthetic peptides predicted from putative complex I subunit genes encoded by mitochondrial and nuclear T. brucei DNA. Blue Native polyacrylamide gel electrophoresis of T. brucei mitochondrial membrane proteins followed by immunoblotting revealed the presence of a putative complex I with a molecular mass of 600 kDa, which contains a minimum of 11 polypeptides determined by second-dimensional Tricine-SDS/PAGE including the 51 kDa, PSST and TYKY subunits.

Animals↗
PubMed1999

Humics as an electron donor for anaerobic respiration.

The possibility that microorganisms might use reduced humic substances (humics) as an electron donor for the reduction of electron acceptors with a more positive redox potential was investigated. All of the Fe(III)- and humics-reducing microorganisms evaluated were capable of oxidizing reduced humics and/or the reduced humics analogue anthrahydroquinone-2,6,-disulphonate (AHODS), with nitrate and/or fumarate as the electron acceptor. These included Geobacter metallireducens, Geobacter sulphurreducens, Geothrix fermentans, Shewanella alga, Wolinella succinogenes and 'S. barnesii'. Several of the humics-oxidizing microorganisms grew in medium with AHQDS as the sole electron donor and fumarate as the electron acceptor. Even though it does not reduce Fe(III) or humics, Paracoccus denitrificans could use AHQDS and reduced humics as electron donors for denitrification. However, another denitrifier, Pseudomonas denitrificans, could not. AHODS could also serve as an electron donor for selenate and arsenate reduction by W. succinogenes. Electron spin resonance studies demonstrated that humics oxidation was associated with the oxidation of hydroquinone moieties in the humics. Studies with G. metallireducens and W. succinogenes demonstrated that the anthraquinone-2,6-disulphonate (AQDS)/AHQDS redox couple mediated an interspecies electron transfer between the two organisms. These results suggest that, as microbially reduced humics enter less reduced zones of soils and sediments, the reduced humics may serve as electron donors for microbial reduction of several environmentally significant electron acceptors.

Aerobiosis↗
PubMed1983

Receptor sites involved in posttranslational transport of apocytochrome c into mitochondria: specificity, affinity, and number of sites.

Assembly of cytochrome c involves a series of steps: synthesis of apocytochrome c on free ribosomes, specific binding of apocytochrome c to the mitochondrial surface, transfer across the outer membrane, covalent addition of protoheme, refolding of the polypeptide chain, and association of holocytochrome c with its functional sites at the inner membrane. The binding step of apocytochrome c to Neurospora crassa mitochondria was studied by inhibiting the subsequent transfer steps with the heme analogue deuterohemin. The binding sites are highly specific for mitochondrial apocytochromes c. Bound labeled Neurospora apocytochrome c was competitively displaced by unlabeled apocytochrome c from various species. These exhibited different abilities for displacement. Apocytochrome c from Paracoccus denitrificans, the amino-terminal (heme-binding) fragment of Neurospora apocytochrome c, and Neurospora holocytochrome c did not recognize the binding sites. Polylysine did not interfere with apocytochrome c binding. Apocytochrome c is reversibly bound. The binding sites are present in limited number. High-affinity binding sites were present at about 90 pmol/mg of mitochondrial protein. They displayed an association constant of 2.2 X 10(7) M-1. Apocytochrome c was imported into mitochondria and converted to holocytochrome c directly from the binding sites when inhibition by deuterohemin was relieved. We conclude that the apocytochrome c binding sites on mitochondria represent receptors that function in the recognition and import of this precursor by mitochondria.

Animals↗
PubMed1997

The roles of the two proton input channels in cytochrome c oxidase from Rhodobacter sphaeroides probed by the effects of site-directed mutations on time-resolved electrogenic intraprotein proton transfer.

The crystal structures of cytochrome c oxidase from both bovine and Paracoccus denitrificans reveal two putative proton input channels that connect the heme-copper center, where dioxygen is reduced, to the internal aqueous phase. In this work we have examined the role of these two channels, looking at the effects of site-directed mutations of residues observed in each of the channels of the cytochrome c oxidase from Rhodobacter sphaeroides. A photoelectric technique was used to monitor the time-resolved electrogenic proton transfer steps associated with the photo-induced reduction of the ferryl-oxo form of heme a3 (Fe4+ = O2-) to the oxidized form (Fe3+OH-). This redox step requires the delivery of a "chemical" H+ to protonate the reduced oxygen atom and is also coupled to proton pumping. It is found that mutations in the K channel (K362M and T359A) have virtually no effect on the ferryl-oxo-to-oxidized (F-to-Ox) transition, although steady-state turnover is severely limited. In contrast, electrogenic proton transfer at this step is strongly suppressed by mutations in the D channel. The results strongly suggest that the functional roles of the two channels are not the separate delivery of chemical or pumped protons, as proposed recently [Iwata, S., Ostermeier, C., Ludwig, B. & Michel, H. (1995) Nature (London) 376, 660-669]. The D channel is likely to be involved in the uptake of both "chemical" and "pumped" protons in the F-to-Ox transition, whereas the K channel is probably idle at this partial reaction and is likely to be used for loading the enzyme with protons at some earlier steps of the catalytic cycle. This conclusion agrees with different redox states of heme a3 in the K362M and E286Q mutants under aerobic steady-state turnover conditions.

Animals↗
PubMed1997

The archaeal SoxABCD complex is a proton pump in Sulfolobus acidocaldarius.

The thermoacidophilic archaeon Sulfolobus acidocaldarius expresses a very unusual quinol oxidase, which contains four heme a redox centers and one copper atom. The enzyme was solubilized with dodecyl maltoside and purified to homogeneity by a combination of hydrophobic interaction and anion exchange chromatography. The oxidase complex consists of four polypeptide subunits with apparent molecular masses of 64, 39, 27, and 14 kDa that are encoded by the soxABCD operon (Lübben, M., Kolmerer, B., and Saraste, M. (1992) EMBO J. 11, 805-812). The optical spectra and redox potentials of the SoxABCD complex have been characterized, and the absorption coefficients of the contributing cytochromes a587 and aa3 were determined. The EPR spectra indicate the presence of three low spin and one high spin heme species, the latter associated with the binuclear heme CuB site. Standard midpoint potentials of the cytochrome a587 heme centers were determined as +210 and +270 mV, respectively. The maximum turnover of the complex (1300 s-1 at 65 degrees C) was found to be about three times greater than that of the previously studied isolated cytochrome aa3 subunit alone (Gleissner, M., Elferink, M. G., Driessen, A. J., Konings, W. N., Anemüller, S., and Schäfer, G. (1994) Eur. J. Biochem. 224, 983-990). With N,N,N',N'-tetramethyl-1,4-phenylenediamine as a reductant, the SoxABCD complex reconstituted into liposomes generates a proton motive force. A new method is described by co-reconstitution of SoxABCD with a Sulfolobus Rieske FeS-protein (SoxL), allowing energization by cytochrome c. It is based on the finding that this Rieske protein can equilibrate electrons between cytochrome c and quinones reversibly (Schmidt, C. L., Anemüller, S., Teixeira, M., and Schäfer, G. (1995) FEBS Lett. 359, 239-243). With this system, generating no scalar protons, the stoichiometry of proton translocation could be determined. A net H+/e- ratio >1 was determined, identifying the SoxABCD complex as a proton-pumping quinol oxidase. According to structural analysis, the cytochrome aa3 moiety of the complex does not contain the signature of a H+ pumping channel as identified in Rhodobacter sphaeroides or Paracoccus denitrificans. Therefore, for H+ translocation, a mechanism different from that in typical heme-copper oxidases of the aa3 or bo3 type is discussed.

Bacterial Outer Membrane Proteins↗
PubMed1997

The proton-translocating NADH-quinone oxidoreductase (NDH-1) of thermophilic bacterium Thermus thermophilus HB-8. Complete DNA sequence of the gene cluster and thermostable properties of the expressed NQO2 subunit.

The genes encoding the proton-translocating NADH-quinone oxidoreductase (NDH-1) of a thermophilic bacterium Thermus thermophilus HB-8 were cloned and sequenced. They constitute a cluster that is composed of 14 structural genes and contains no unidentified reading frames. All of the 14 structural genes, which are designated NQO1-14, encode subunits homologous to those of Paracoccus denitrificans NDH-1, respectively, and are arranged in the same order as other bacterial NDH-1 genes. T. thermophilus NDH-1 contains at most nine putative iron-sulfur cluster binding sites, eight of which are commonly found in other organisms. The T. thermophilus NQO2 subunit was expressed in Escherichia coli. The expressed subunit bears a single [2Fe-2S] cluster whose optical and EPR properties are very similar to those of N1a cluster in the P. denitrificans NQO2 subunit (Yano, T., Sled', V.D., Ohnishi, T., and Yagi, T. (1994) Biochemistry 33, 494-499). These results strongly suggest that the T. thermophilus NDH-1 is similar to other NDH-1 enzyme complexes in terms of subunit and cofactor composition. The T. thermophilus NQO2 subunit displayed much higher stability than the mesophilic equivalent and its iron-sulfur cluster remained intact even after incubation for 3 h at 65 degrees C under anaerobic conditions. With the advantage of thermostability, the T. thermophilus NDH-1 provides a great model system to investigate the structure-function relationship of the NDH-1 enzyme complexes.

Amino Acid Sequence↗
PubMed1998

Crystallographic and spectroscopic studies of native, aminoquinol, and monovalent cation-bound forms of methylamine dehydrogenase from Methylobacterium extorquens AM1.

Various monovalent cations influence the enzymatic activity and the spectroscopic properties of methylamine dehydrogenase (MADH). Here, we report the structure determination of this tryptophan tryptophylquinone-containing enzyme from Methylobacterium extorquens AM1 by high resolution x-ray crystallography (1.75 A). This first MADH crystal structure at low ionic strength is compared with the high resolution structure of the related MADH from Paracoccus denitrificans recently reported. We also describe the first structures (at 1.95 to 2.15 A resolution) of an MADH in the substrate-reduced form and in the presence of trimethylamine and of cesium, two competitive inhibitors. Polarized absorption microspectrophotometry was performed on single crystals under various redox, pH, and salt conditions. The results show that the enzyme is catalytically active in the crystal and that the cations cause the same spectral perturbations as are observed in solution. These studies lead us to propose a model for the entrance and binding of the substrate in the active site.

Bacterial Proteins↗
PubMed1999

The structure of an electron transfer complex containing a cytochrome c and a peroxidase.

Efficient biological electron transfer may require a fluid association of redox partners. Two noncrystallographic methods (a new molecular docking program and 1H NMR spectroscopy) have been used to study the electron transfer complex formed between the cytochrome c peroxidase (CCP) of Paracoccus denitrificans and cytochromes c. For the natural redox partner, cytochrome c550, the results are consistent with a complex in which the heme of a single cytochrome lies above the exposed electron-transferring heme of the peroxidase. In contrast, two molecules of the nonphysiological but kinetically competent horse cytochrome bind between the two hemes of the peroxidase. These dramatically different patterns are consistent with a redox active surface on the peroxidase that may accommodate more than one cytochrome and allow lateral mobility.

Animals↗
PubMed1999

Does the reduction of c heme trigger the conformational change of crystalline nitrite reductase?

The structures of nitrite reductase from Paracoccus denitrificans GB17 (NiR-Pd) and Pseudomonas aeruginosa (NiR-Pa) have been described for the oxidized and reduced state (Fülöp, V., Moir, J. W. B., Ferguson, S. J., and Hajdu, J. (1995) Cell 81, 369-377; Nurizzo, D., Silvestrini, M. C., Mathieu, M., Cutruzzolà, F., Bourgeois, D., Fülöp, V., Hajdu, J., Brunori, M., Tegoni, M., and Cambillau, C. (1997) Structure 5, 1157-1171; Nurizzo, D., Cutruzzolà, F., Arese, M., Bourgeois, D., Brunori, M., Cambillau, C. , and Tegoni, M. (1998) Biochemistry 37, 13987-13996). Major conformational rearrangements are observed in the extreme states although they are more substantial in NiR-Pd. The four structures differ significantly in the c heme domains. Upon reduction, a His17/Met106 heme-ligand switch is observed in NiR-Pd together with concerted movements of the Tyr in the distal site of the d1 heme (Tyr10 in NiR-Pa, Tyr25 in NiR-Pd) and of a loop of the c heme domain (56-62 in NiR-Pa, 99-116 in NiR-Pd). Whether the reduction of the c heme, which undergoes the major rearrangements, is the trigger of these movements is the question addressed by our study. This conformational reorganization is not observed in the partially reduced species, in which the c heme is partially or largely (15-90%) reduced but the d1 heme is still oxidized. These results suggest that the d1 heme reduction is likely to be responsible of the movements. We speculate about the mechanistic explanation as to why the opening of the d1 heme distal pocket only occurs upon electron transfer to the d1 heme itself, to allow binding of the physiological substrate NO2- exclusively to the reduced metal center.

Ascorbic Acid↗
PubMed2002

Nitric-oxide reductase. Structure and properties of the catalytic site from resonance Raman scattering.

We have applied resonance Raman spectroscopy to investigate the properties of the dinuclear center of oxidized, reduced, and NO-bound nitric-oxide reductase from Paracoccus denitrificans. The spectra of the oxidized enzyme show two distinct nu(as)(Fe-O-Fe) modes at 815 and 833 cm(-1) of the heme/non-heme diiron center. The splitting of the Fe-O-Fe mode suggests that two different conformations (open and closed) are present in the catalytic site of the enzyme. We find evidence from deuterium exchange experiments that in the dominant conformation (833 cm(-1) mode, closed), the Fe-O-Fe unit is hydrogen-bonded to distal residue(s). The ferric nitrosyl complex of nitric-oxide reductase exhibits the nu(Fe(3+)-NO) and nu(N-O) at 594 and 1904 cm(-1), respectively. The nitrosyl species we detect is photolabile and can be photolyzed to generate a new form of oxidized enzyme in which the proximal histidine is ligated to heme b(3), in contrast to the resting form. Photodissociation of the NO ligand yields a five-coordinate high-spin heme b(3). Based on the findings reported here, the structure and properties of the dinuclear center of nitric- oxide reductase in the oxidized, reduced, and NO-bound form as well as its photoproduct can be described with certainty.

Catalytic Domain↗
PubMed2002

A novel type of nitric-oxide reductase. Escherichia coli flavorubredoxin.

Escherichia coli flavorubredoxin is a member of the family of the A-type flavoproteins, which are built by two core domains: a metallo-beta-lactamase-like domain, at the N-terminal region, harboring a non-heme di-iron site, and a flavodoxin-like domain, containing one FMN moiety. The enzyme from E. coli has an extra module at the C terminus, containing a rubredoxin-like center. The A-type flavoproteins are widespread among strict and facultative anaerobes, as deduced from the analysis of the complete prokaryotic genomes. In this report we showed that the recombinant enzyme purified from E. coli has nitric-oxide reductase activity with a turnover number of approximately 15 mol of NO.mol enzyme(-1).s(-1), which was well within the range of those determined for the canonical heme b(3)-Fe(B) containing nitric-oxide reductases (e.g. approximately 10-50 mol NO.mol enzyme(-1).s(-1) for the Paracoccus denitrificans NOR). Furthermore, it was shown that the activity was due to the A-type flavoprotein core, as the rubredoxin domain alone exhibited no activity. Thus, a novel family of prokaryotic NO reductases, with a non-heme di-iron site as the catalytic center, was established.

Base Sequence↗
PubMed2003

Cytochrome c551 from Starkeya novella: characterization, spectroscopic properties, and phylogeny of a diheme protein of the SoxAX family.

Cytochromes from the SoxAX family have a major role in thiosulfate oxidation via the thiosulfate-oxidizing multi-enzyme system (TOMES). Previously characterized SoxAX proteins from Rhodovulum sulfidophilum and Paracoccus pantotrophus contain three heme c groups, two of which are located on the SoxA subunit. In contrast, the SoxAX protein purified from Starkeya novella was found to contain only two heme groups. Mass spectrometry showed that a disulfide bond replaced the second heme group found in the diheme SoxA subunits. Apparent molecular masses of 27,229 +/- 10.3 Da and 20,258.6 +/- 1 Da were determined for SoxA and SoxX with an overall mass of 49.7 kDa, indicating a heterodimeric structure. Optical redox potentiometry found that the two heme cofactors are reduced at similar potentials (versus NHE) that are as follows: +133 mV (pH 6.0); +104 mV (pH 7.0); +49 (pH 7.9) and +10 mV (pH 8.7). EPR spectroscopy revealed that both ferric heme groups are in the low spin state, and the spectra were consistent with one heme having a His/Cys axial ligation and the other having a His/Met axial ligation. The His/Cys ligated heme is present in different conformational states and gives rise to three distinct signals. Amino acid sequencing was used to unambiguously assign the protein to the encoding genes, soxAX, which are part of a complete sox gene cluster found in S. novella. Phylogenetic analysis of soxA- and soxX-related gene sequences indicates a parallel development of SoxA and SoxX, with the diheme and monoheme SoxA sequences located on clearly separated branches of a phylogenetic tree.

Alphaproteobacteria↗
PubMed2003

A superfamily of voltage-gated sodium channels in bacteria.

NaChBac, a six-alpha-helical transmembrane-spanning protein cloned from Bacillus halodurans, is the first functionally characterized bacterial voltage-gated Na(+)-selective channel. As a highly expressing ion channel protein, NaChBac is an ideal candidate for high resolution structural determination and structure-function studies. The biological role of NaChBac, however, is still unknown. In this report, another 11 structurally related bacterial proteins are described. Two of these functionally expressed as voltage-dependent Na(+) channels (Na(V)PZ from Paracoccus zeaxanthinifaciens and Na(V)SP from Silicibacter pomeroyi). Na(V)PZ and Na(V)SP share approximately 40% amino acid sequence identity with NaChBac. When expressed in mammalian cell lines, both Na(V)PZ and Na(V)SP were Na(+)-selective and voltage-dependent. However, their kinetics and voltage dependence differ significantly. These single six-alpha-helical transmembrane-spanning subunits constitute a widely distributed superfamily (Na(V)Bac) of channels in bacteria, implying a fundamental prokaryotic function. The degree of sequence homology (22-54%) is optimal for future comparisons of Na(V)Bac structure and function of similarity and dissimilarity among Na(V)Bac proteins. Thus, the Na(V)Bac superfamily is fertile ground for crystallographic, electrophysiological, and microbiological studies.

Amino Acid Sequence↗
PubMed2004

Functional roles of four conserved charged residues in the membrane domain subunit NuoA of the proton-translocating NADH-quinone oxidoreductase from Escherichia coli.

The H(+)(Na(+))-translocating NADH-quinone (Q) oxidoreductase (NDH-1) of Escherichia coli is composed of 13 different subunits (NuoA-N). Subunit NuoA (ND3, Nqo7) is one of the seven membrane domain subunits that are considered to be involved in H(+)(Na(+)) translocation. We demonstrated that in the Paracoccus denitrificans NDH-1 subunit, Nqo7 (ND3) directly interacts with peripheral subunits Nqo6 (PSST) and Nqo4 (49 kDa) by using cross-linkers (Di Bernardo, S., and Yagi, T. (2001) FEBS Lett. 508, 385-388 and Kao, M.-C., Matsuno-Yagi, A., and Yagi, T. (2004) Biochemistry 43, 3750-3755). To investigate the structural and functional roles of conserved charged amino acid residues, a nuoA knock-out mutant and site-specific mutants K46A, E51A, D79N, D79A, E81Q, E81A, and D79N/E81Q were constructed by utilizing chromosomal DNA manipulation. In terms of immunochemical and NADH dehydrogenase activity-staining analyses, all site-specific mutants are similar to the wild type, suggesting that those NuoA site-specific mutations do not significantly affect the assembly of peripheral subunits in situ. In addition, site-specific mutants showed similar deamino-NADH-K(3)Fe(CN)(6) reductase activity to the wild type. The K46A mutation scarcely inhibited deamino-NADH-Q reductase activity. In contrast, E51A, D79A, D79N, E81A, and E81Q mutation partially suppressed deamino-NADH-Q reductase activity to 30, 90, 40, 40, and 50%, respectively. The double mutant D79N/E81Q almost completely lost the energy-transducing NDH-1 activities but did not display any loss of deamino-NADH-K(3)Fe(CN)(6) reductase activity. The possible functional roles of residues Asp-79 and Glu-81 were discussed.

Amino Acid Sequence↗
PubMed2006

Spectropotentiometric and structural analysis of the periplasmic nitrate reductase from Escherichia coli.

The Escherichia coli NapA (periplasmic nitrate reductase) contains a [4Fe-4S] cluster and a Mo-bis-molybdopterin guanine dinucleotide cofactor. The NapA holoenzyme associates with a di-heme c-type cytochrome redox partner (NapB). These proteins have been purified and studied by spectropotentiometry, and the structure of NapA has been determined. In contrast to the well characterized heterodimeric NapAB systems ofalpha-proteobacteria, such as Rhodobacter sphaeroides and Paracoccus pantotrophus, the gamma-proteobacterial E. coli NapA and NapB proteins purify independently and not as a tight heterodimeric complex. This relatively weak interaction is reflected in dissociation constants of 15 and 32 mum determined for oxidized and reduced NapAB complexes, respectively. The surface electrostatic potential of E. coli NapA in the apparent NapB binding region is markedly less polar and anionic than that of the alpha-proteobacterial NapA, which may underlie the weaker binding of NapB. The molybdenum ion coordination sphere of E. coli NapA includes two molybdopterin guanine dinucleotide dithiolenes, a protein-derived cysteinyl ligand and an oxygen atom. The Mo-O bond length is 2.6 A, which is indicative of a water ligand. The potential range over which the Mo(6+) state is reduced to the Mo(5+) state in either NapA (between +100 and -100 mV) or the NapAB complex (-150 to -350 mV) is much lower than that reported for R. sphaeroides NapA (midpoint potential Mo(6+/5+) > +350 mV), and the form of the Mo(5+) EPR signal is quite distinct. In E. coli NapA or NapAB, the Mo(5+) state could not be further reduced to Mo(4+). We then propose a catalytic cycle for E. coli NapA in which nitrate binds to the Mo(5+) ion and where a stable des-oxo Mo(6+) species may participate.

Crystallography, X-Ray↗