Nitric oxide signaling and transcriptional control of denitrification genes in Pseudomonas stutzeri.

The expression of denitrification by a facultatively anaerobic bacterium requires as exogenous signals a low oxygen tension concomitant with an N oxide. We have studied the role of nitric oxide (NO), nitrous oxide (N2O), and nitrite as signal molecules for the expression of the denitrification apparatus of Pseudomonas stutzeri. Transcriptional kinetics of structural genes were monitored by Northern blot analysis in a 60-min time frame after cells were exposed to an N oxide signal. To differentiate the inducer role of NO from that of nitrite, mRNA kinetics were monitored under anoxic conditions in a nirF strain, where NO generation from nitrite is prevented because of a defect in heme D(1) biosynthesis. NO-triggered responses were monitored from the nirSTB operon (encoding cytochrome cd(1) nitrite reductase), the norCB operon (encoding NO reductase), nosZ (encoding nitrous oxide reductase), and nosR (encoding a putative regulator). Transcription of nirSTB and norCB was activated by 5 to 50 nM NO, whereas the nosZ promoter required about 250 nM. Nitrite at 5 to 50 nM elicited no response. At a threshold concentration of 650 nM N2O, we observed in the anoxic cell the transient appearance of nosZ and nosR transcripts. Constant levels of transcripts of both genes were observed in an anoxic cell sparged with N2O. NO at 250 nM stimulated in this cell type the expression of nos genes severalfold. The transcription factor DnrD, a member of the FNR-CRP family, was found to be part of the NO-triggered signal transduction pathway. However, overexpression of dnrD in an engineered strain did not result in NirS synthesis, indicating a need for activation of DnrD. NO modified the transcriptional pattern of the dnrD operon by inducing the transcription of dnrN and dnrO, located upstream of dnrD. Insertional mutagenesis of dnrN altered the kinetic response of the nirSTB operon towards nitrite. Our data establish NO and DnrD as key elements in the regulatory network of denitrification in P. stutzeri. The NO response adds to the previously identified nitrate-nitrite response mediated by the NarXL two-component system for the expression of respiratory nitrate reductase encoded by the narGHJI operon.

Kinetics of CO binding and CO photodissociation in Pseudomonas stutzeri cd(1) nitrite reductase: probing the role of extended N-termini in fast structural relaxation upon CO photodissociation.

cd(1) nitrite reductase from Pseudomonas stutzeri is a di-haem- containing enzyme, comprising a c-type haem and a d-type haem. Studies with the highly related cd(1) nitrite reductase of Pseudomonas aeruginosa have established that this enzyme undergoes fast (microsecond) and global structural relaxation upon CO photodissociation from the reduced enzyme. A key difference between the Ps. aeruginosa and Ps. stutzeri enzyme is the absence of a flexible N-terminal extension in the Ps. stutzeri enzyme. In Ps. aeruginosa cd(1) nitrite reductase the N-terminal extension wraps around the second subunit of the homodimer and with Tyr(10) stabilizing a water molecule co-ordinated to the d(1)-haem. Given the intimate association of the N-terminal extension with the d(1)-haem, we hypothesized that the presence of the N-terminal extension likely contributes to the fast structural reorganization seen during photodissociation of CO from the reduced enzyme. In the present study we have investigated the kinetics of CO association and CO photodissociation of Ps. stutzeri cd(1) nitrite reductase (which lacks the N-terminal arm seen in the Ps. aeruginosa enzyme) to probe the role and influence of the N-terminal arm in the fast global structural reorganization seen with Ps. aeruginosa. Surprisingly, we find that Ps. stutzeri cd(1) nitrite reductase also undergoes fast structural reorganization during CO photodissociation. We also show, in stopped-flow experiments, that the kinetics of CO binding and dissociation with reduced Ps. stutzeri cd(1) nitrite reductase are similar to those observed with Ps. aeruginosa enzyme, thus ruling out a major role for the N-terminal flexible arm found in Ps. aeruginosa in the kinetics of these processes. Our data indicate that global structural reorganization following CO photodissociation is an intrinsic property of the haem domains in cd(1) nitrite reductases. The absence of an N-terminal extension, as in the Ps. stutzeri cd(1) nitrite reductase, does not lead to loss of global structural reorganization following CO photodissociation.

Role of the Tat ransport system in nitrous oxide reductase translocation and cytochrome cd1 biosynthesis in Pseudomonas stutzeri.

By transforming N2O to N2, the multicopper enzyme nitrous oxide reductase provides a periplasmic electron sink for a respiratory chain that is part of denitrification. The signal sequence of the enzyme carries the heptameric twin-arginine consensus motif characteristic of the Tat pathway. We have identified tat genes of Pseudomonas stutzeri and functionally analyzed the unlinked tatC and tatE loci. A tatC mutant retained N2O reductase in the cytoplasm in the unprocessed form and lacking the metal cofactors. This is contrary to viewing the Tat system as specific only for fully assembled proteins. A C618V exchange in the electron transfer center CuA rendered the enzyme largely incompetent for transport. The location of the mutation in the C-terminal domain of N(2)O reductase implies that the Tat system acts on a completely synthesized protein and is sensitive to a late structural variation in folding. By generating a tatE mutant and a reductase-overproducing strain, we show a function for TatE in N2O reductase translocation. Further, we have found that the Tat and Sec pathways have to cooperate to produce a functional nitrite reductase system. The cytochrome cd1 nitrite reductase was found in the periplasm of the tatC mutant, suggesting export by the Sec pathway; however, the enzyme lacked the heme D1 macrocycle. The NirD protein as part of a complex required for heme D1 synthesis or processing carries a putative Tat signal peptide. Since NO reduction was also inhibited in the tatC mutant, the Tat protein translocation system is necessary in multiple ways for establishing anaerobic nitrite denitrification.

The catalytic center in nitrous oxide reductase, CuZ, is a copper-sulfide cluster.

The crystal structure of nitrous oxide reductase, the enzyme catalyzing the final step of bacterial denitrification in which nitrous oxide is reduced to dinitrogen, exhibits a novel catalytic site, called Cu(Z). This comprises a cluster of four copper ions bound by seven histidines and three other ligands modeled in the X-ray structure as OH(-) or H(2)O. However, elemental analyses and resonance Raman spectroscopy of isotopically labeled enzyme conclusively demonstrate that Cu(Z) has one acid-labile sulfur ligand. Thus, nitrous oxide reductase contains the first reported biological copper-sulfide cluster.

Structural investigations of the CuA centre of nitrous oxide reductase from Pseudomonas stutzeri by site-directed mutagenesis and X-ray absorption spectroscopy.

Nitrous oxide reductase is the terminal component of a respiratory chain that utilizes N2O in lieu of oxygen. It is a homodimer carrying in each subunit the electron transfer site, CuA, and the substrate-reducing catalytic centre, CuZ. Spectroscopic data have provided robust evidence for CuA as a binuclear, mixed-valence metal site. To provide further structural information on the CuA centre of N2O reductase, site directed mutagenesis and Cu K-edge X-ray absorption spectroscopic investigation have been undertaken. Candidate amino acids as ligands for the CuA centre of the enzyme from Pseudomonas stutzeri ATCC14405 were substituted by evolutionary conserved residues or amino acids similar to the wild-type residues. The mutations identified the amino acids His583, Cys618, Cys622 and Met629 as ligands of Cu1, and Cys618, Cys622 and His626 as the minimal set of ligands for Cu2 of the CuA centre. Other amino acid substitutions indicated His494 as a likely ligand of CuZ, and an indirect role for Asp580, compatible with a docking function for the electron donor. Cu binding and spectroscopic properties of recombinant N2O reductase proteins point at intersubunit or interdomain interaction of CuA and CuZ. Cu K-edge X-ray absorption spectra have been recorded to investigate the local environment of the Cu centres in N2O reductase. Cu K-edge Extended X-ray Absorption Fine Structure (EXAFS) for binuclear Cu chemical systems show clear evidence for Cu backscattering at approximately 2.5 A. The Cu K-edge EXAFS of the CuA centre of N2O reductase is very similar to that of the CuA centre of cytochrome c oxidase and the optimum simulation of the experimental data involves backscattering from a histidine group with Cu-N of 1.92 A, two sulfur atoms at 2.24 A and a Cu atom at 2. 43 A, and allows for the presence of a further light atom (oxygen or nitrogen) at 2.05 A. The interpretation of the CuA EXAFS is in line with ligands assigned by site-directed mutagenesis. By a difference spectrum approach, using the Cu K-edge EXAFS of the holoenzyme and that of the CuA-only form, histidine was identified as a major contributor to the backscattering. A structural model for the CuA centre of N2O reductase has been generated on the basis of the atomic coordinates for the homologous domain of cytochrome c oxidase and incorporating our current results and previous spectroscopic data.

The heme-copper oxidases of Thermus thermophilus catalyze the reduction of nitric oxide: evolutionary implications.

We show that the heme-copper terminal oxidases of Thermus thermophilus (called ba(3) and caa(3)) are able to catalyze the reduction of nitric oxide (NO) to nitrous oxide (N(2)O) under reducing anaerobic conditions. The rate of NO consumption and N(2)O production were found to be linearly dependent on enzyme concentration, and activity was abolished by enzyme denaturation. Thus, contrary to the eukaryotic enzyme, both T. thermophilus oxidases display a NO reductase activity (3.0 +/- 0.7 mol NO/mol ba(3) x min and 32 +/- 8 mol NO/mol caa(3) x min at [NO] approximately 50 microM and 20 degrees C) that, though considerably lower than that of bona fide NO reductases (300-4,500 mol NO/mol enzyme x min), is definitely significant. We also show that for ba(3) oxidase, NO reduction is associated to oxidation of cytochrome b at a rate compatible with turnover, suggesting a mechanism consistent with the stoichiometry of the overall reaction. We propose that the NO reductase activity of T. thermophilus oxidases may depend on a peculiar Cu(B)(+) coordination, which may be revealed by the forthcoming three-dimensional structure. These findings support the hypothesis of a common phylogeny of aerobic respiration and bacterial denitrification, which was proposed on the basis of structural similarities between the Pseudomonas stutzeri NO reductase and the cbb(3) terminal oxidases. Our findings represent functional evidence in support of this hypothesis.

1H NMR studies on the CuA center of nitrous oxide reductase from Pseudomonas stutzeri.

1H NMR spectra of the CuA center of N2OR from Pseudomonas stutzeri, and a mutant enzyme that contains only CuA, were recorded in both H2O- and D2O-buffered solution at pH 7.5. Several sharp, well-resolved hyperfine-shifted 1H NMR signals were observed in the 60 to -10 ppm chemical shift range. Comparison of the native and mutant N2OR spectra recorded in H2O-buffered solutions indicated that several additional signals are present in the native protein spectrum. These signals are attributed to a dinuclear copperII center. At least two of the observed hyperfine-shifted signals associated with the dinuclear center, those at 23.0 and 13.2 ppm, are lost upon replacement of H2O buffer with D2O buffer. These data indicate that at least two histidine residues are ligands of a dinuclear CuII center. Comparison of the mutant N2OR 1H NMR spectra recorded in H2O and D2O indicates that three signals, c (27.5 ppm), e (23.6 ppm), and i (12.4 ppm), are solvent exchangeable. The two most strongly downfield-shifted signals (c and e) are assigned to the two N epsilon 2H (N-H) protons of the coordinated histidine residues, while the remaining exchangeable signal is assigned to a backbone N-H proton in close proximity to the CuA cluster. Signal e was found to decrease in intensity as the temperature was increased, indicating that proton e resides on a more solvent-exposed histidine residue. One-dimensional nOe studies at pH 7.5 allowed the histidine ring protons to be definitively assigned, while the remaining signals were assigned by comparison to previously reported spectra from CuA centers. The temperature dependence of the observed hyperfine-shifted 1H NMR signals of mutant N2OR were recorded over the temperature range of 276-315 K. Both Curie and anti-Curie temperature dependencies are observed for sets of hyperfine-shifted protons. Signals a and h (cysteine protons) follow anti-Curie behavior (contact shift increases with increasing temperatures), while signals b-g, i, and j (histidine protons) follow Curie behavior (contact shift decreases with increasing temperatures). Fits of the temperature dependence of the observed hyperfine-shifted signals provided the energy separation (Delta EL) between the ground (2B3u) and excited (2B2u) states. The temperature data obtained for all of the observed hyperfine-shifted histidine ligand protons provided a Delta EL value of 62 +/- 35 cm-1. The temperature dependence of the observed cysteine C beta H and C alpha H protons (a and h) were fit in a separate experiment providing a Delta EL value of 585 +/- 125 cm-1. The differences between the Delta EL values determined by 1H NMR spectroscopy and those determined by EPR or MCD likely arise from coupling between relatively low-frequency vibrational states and the ground and excited electronic states.

Nitrate and nitrite control of respiratory nitrate reduction in denitrifying Pseudomonas stutzeri by a two-component regulatory system homologous to NarXL of Escherichia coli.

Bacterial denitrification is expressed in response to the concurrent exogenous signals of low-oxygen tension and nitrate or one of its reduction products. The mechanism by which nitrate-dependent gene activation is effected was investigated in the denitrifying bacterium Pseudomonas stutzeri ATCC 14405. We have identified and isolated from this organism the chromosomal region encoding the two-component sensor-regulator pair NarXL and found that it is linked with the narG operon for respiratory nitrate reductase. The same region encodes two putative nitrate or nitrite translocases, NarK and NarC (the latter shows the highest similarity to yeast [Pichia] and plant [Nicotiana] nitrate transporters), and the nitrate-regulated transcription factor, DnrE, of the FNR family. The roles of NarX and NarL in nitrate respiration were studied with deletion mutants. NarL activated the transcription of narG, narK, and dnrE but did not affect the denitrification regulons for the respiratory substrates nitrite, nitric oxide, and nitrous oxide. The promoters of narG, narK, and dnrE carry sequence motifs, TACYYMT, which correspond to the NarL recognition sequence established for Escherichia coli. The cellular response toward nitrate and nitrite was mediated by the sensor protein NarX, which discriminated weakly between these oxyanions. Our data show that the NarXL two-component regulatory system has been incorporated into the bacterial denitrification process of P. stutzeri for selective regulation of nitrate respiration.

Multiple transcription factors of the FNR family in denitrifying Pseudomonas stutzeri: characterization of four fnr-like genes, regulatory responses and cognate metabolic processes.

Pseudomonas stutzeri is a facultative anaerobic bacterium with the capability of denitrification. In searching for regulators that control the expression of this trait in response to oxygen withdrawal, we have found an unprecedented multiplicity of four genes encoding transcription factors of the FNR family. The fnrA gene encodes a genuine FNR-type regulator, which is expressed constitutively and controls the cytochrome cbb3-type terminal oxidase (the cco operon), cytochrome c peroxidase (the ccp gene) and the oxygen-independent coproporphyrinogen III oxidase (the hemN gene), in addition to its previously demonstrated role in arginine catabolism (the arc operon). The fnr homologues dnrD, dnrE and dnrS encode regulators of a new subgroup within the FNR family. Their main distinctive feature is the lack of cysteine residues for complexing the [4Fe-4S] centre of redox-active FNR-type regulators. However, they form a phylogenetic lineage separate from the FixK branch of FNR proteins, which also lack this cysteine signature. We have studied the expression of the dnr genes under aerobic, oxygen-limited and denitrifying conditions. DnrD is a key regulator of denitrification by selective activation of the genes for cytochrome cd1 nitrite reductase and NO reductase. The dnrD gene is part of the 30 kb region carrying denitrification genes of P. stutzeri. Transcription of dnrD was activated in O2-limited cells and particularly strongly in denitrifying cells, but was not under the control of FnrA. In response to denitrifying growth conditions, dnrD was transcribed as part of an operon together with genes downstream and upstream of dnrD. dnrS was found about 9 kb upstream of dnrD, next to the nrdD gene for anaerobic ribonucleotide reductase. The transcription of dnrS required FnrA in O2-limited cells. Mutation of dnrS affected nrdD and the expression of ferredoxin I as an element of the oxidative stress response. The dnrE gene is part of the nar region encoding functions for respiratory nitrate reduction. We found the highest amount of dnrE transcripts in aerobically nitrate-challenged cells. The gene was transcribed from two promoters, P1 and P2, of which promoter P1 was under the control of the nitrate response regulator NarL. The multiplicity of FNR factors in P. stutzeri underlines the versatility of the FNR scaffold to serve for transcriptional regulation directed at anaerobic or nitrate-activated metabolic processes.

Kinetics of nirS expression (cytochrome cd1 nitrite reductase) in Pseudomonas stutzeri during the transition from aerobic respiration to denitrification: evidence for a denitrification-specific nitrate- and nitrite-responsive regulatory system.

After shifting an oxygen-respiring culture of Pseudomonas stutzeri to nitrate or nitrite respiration, we directly monitored the expression of the nirS gene by mRNA analysis. nirS encodes the 62-kDa subunit of the homodimeric cytochrome cd1 nitrite reductase involved in denitrification. Information was sought about the requirements for gene activation, potential regulators of such activation, and signal transduction pathways triggered by the alternative respiratory substrates. We found that nirS, together with nirT and nirB (which encode tetra- and diheme cytochromes, respectively), is part of a 3.4-kb operon. In addition, we found a 2-kb monocistronic transcript. The half-life of each of these messages was approximately 13 min in denitrifying cells with a doubling time of around 2.5 h. When the culture was subjected to a low oxygen tension, we observed a transient expression of nirS lasting for about 30 min. The continued transcription of the nirS operon required the presence of nitrate or nitrite. This anaerobically manifested N-oxide response was maintained in nitrate sensor (NarX) and response regulator (NarL) knockout strains. Similar mRNA stability and transition kinetics were observed for the norCB operon, encoding the NO reductase complex, and the nosZ gene, encoding nitrous oxide reductase. Our results suggest that a nitrate- and nitrite-responsive regulatory circuit independent of NarXL is necessary for the activation of denitrification genes.

CuA and CuZ are variants of the electron transfer center in nitrous oxide reductase.

Nitrous oxide reductase (N2OR) is a dimeric copper-dependent bacterial enzyme that catalyzes the reduction of N2O to N2 as part of the denitrification pathway. In the absence of an x-ray crystal structure, the current model of the nature of the copper sites within the enzyme is based on four copper atoms per monomer and assigns two copper atoms to an electron transfer center, CuA, a bis-thiolate-bridged dinuclear copper center found to date only in N2OR and cytochrome c oxidase, and two copper atoms to a second dinuclear center, CuZ, presumed to be the site of catalysis. Based on detailed analysis of the low temperature magnetic CD spectra of N2OR, this paper revises the current model and proposes that both CuA and CuZ are variants of an electron transfer center and hence that all of the observed optical features are due to this electron transfer center. It is proposed further that the presence of these different forms provides a mechanism for the delivery of two electrons to an active site comprising copper ions lacking thiolate coordination.

The requirement of RpoN (sigma factor sigma54) in denitrification by Pseudomonas stutzeri is indirect and restricted to the reduction of nitrite and nitric oxide.

The rpoN region of Pseudomonas stutzeri was cloned, and an rpoN null mutant was constructed. RpoN was not essential for denitrification in this bacterium but affected the expression levels and enzymatic activities of cytochrome cd1 nitrite reductase and nitric oxide reductase, whereas those of respiratory nitrate reductase and nitrous oxide reductase were comparable to wild-type levels. Since the transcription of the structural genes nirS and norCB, coding for nitrite reductase and the nitric oxide reductase complex, respectively, proceeded unabated, our data indicate a posttranslational process for the two key enzymes of denitrification depending on RpoN.

The MCD and EPR of the heme centers of nitric oxide reductase from Pseudomonas stutzeri: evidence that the enzyme is structurally related to the heme-copper oxidases.

EPR spectra at liquid helium temperatures and MCD spectra at room temperature and 4.2 K are presented for fully oxidized nitric oxide reductase (NOR) from Pseudomonas stutzeri. The MCD spectra show that the enzyme contains three heme groups at equivalent concentrations but distinctive in their axial coordination. Two, in the low-spin ferric state at all temperatures, give rise to infrared charge-transfer transitions which show the hemes to have bis-histidine and histidine-methionine ligation, respectively. The EPR spectra show them to be magnetically isolated. The third heme has an unusual temperature-dependent spin state and spectroscopic features which are consistent with histidine-hydroxide coordination. No EPR signals have been detected from this heme. Together with its unusual near-infrared MCD, this suggests a spin-spin interaction between this heme and another paramagnet. The three hemes account for only 75% of the iron content, and it is concluded that the additional paramagnet is a mononuclear ferric ion. These results provide further evidence that NOR is indeed structurally related to heme-copper oxidases and that it contains a heme/non-heme iron spin-coupled pair at the active site.

Cell biology and molecular basis of denitrification.

Denitrification is a distinct means of energy conservation, making use of N oxides as terminal electron acceptors for cellular bioenergetics under anaerobic, microaerophilic, and occasionally aerobic conditions. The process is an essential branch of the global N cycle, reversing dinitrogen fixation, and is associated with chemolithotrophic, phototrophic, diazotrophic, or organotrophic metabolism but generally not with obligately anaerobic life. Discovered more than a century ago and believed to be exclusively a bacterial trait, denitrification has now been found in halophilic and hyperthermophilic archaea and in the mitochondria of fungi, raising evolutionarily intriguing vistas. Important advances in the biochemical characterization of denitrification and the underlying genetics have been achieved with Pseudomonas stutzeri, Pseudomonas aeruginosa, Paracoccus denitrificans, Ralstonia eutropha, and Rhodobacter sphaeroides. Pseudomonads represent one of the largest assemblies of the denitrifying bacteria within a single genus, favoring their use as model organisms. Around 50 genes are required within a single bacterium to encode the core structures of the denitrification apparatus. Much of the denitrification process of gram-negative bacteria has been found confined to the periplasm, whereas the topology and enzymology of the gram-positive bacteria are less well established. The activation and enzymatic transformation of N oxides is based on the redox chemistry of Fe, Cu, and Mo. Biochemical breakthroughs have included the X-ray structures of the two types of respiratory nitrite reductases and the isolation of the novel enzymes nitric oxide reductase and nitrous oxide reductase, as well as their structural characterization by indirect spectroscopic means. This revealed unexpected relationships among denitrification enzymes and respiratory oxygen reductases. Denitrification is intimately related to fundamental cellular processes that include primary and secondary transport, protein translocation, cytochrome c biogenesis, anaerobic gene regulation, metalloprotein assembly, and the biosynthesis of the cofactors molybdopterin and heme D1. An important class of regulators for the anaerobic expression of the denitrification apparatus are transcription factors of the greater FNR family. Nitrate and nitric oxide, in addition to being respiratory substrates, have been identified as signaling molecules for the induction of distinct N oxide-metabolizing enzymes.

Lack of copper insertion into unprocessed cytoplasmic nitrous oxide reductase generated by an R20D substitution in the arginine consensus motif of the signal peptide.

Metal insertion into an engineered cytoplasmic form of the multicopper enzyme N2O reductase (N2OR) (EC 1.7.99.6) of Pseudomonas stutzeri was studied. The reductase has an unusually long presequence of 50 amino acids for translocation into the periplasm. The signal peptide of N2OR shares a conserved twin-arginine sequence motif with the signal peptides of other N2O reductases and a sizeable group of periplasmic or membrane-bound enzymes, requiring cofactor insertion or processing. A catalytically inactive reductase, N2ORR20D, that lacked Cu, accumulated in the cytoplasm on mutation of the first arginine of this motif. The CuA site of N2ORR20D could be reconstituted in vitro indicating that the lack of metal was not due to a serious conformational restraint. Our findings locate the event of in vivo Cu insertion into N2OR in the periplasm or allow it to take place concomitant with protein translocation.

Enzyme diversity and mosaic gene organization in denitrification.

Denitrification is a main branch of the global nitrogen cycle. In the past ten years unravelling the underlying biochemistry and genetics has proceeded at an increasing pace. Fungal denitrification has become a new field. The biochemical investigation of denitrification has culminated in the description of the crystal structures of the two types of nitrite reductases. The N2O reductase shares with cytochrome c oxidase the CuA center as a structurally novel metal site. The cytochrome b subunit of NO reductase has a striking conservation of heme-binding transmembrane segments versus the subunit I of cytochrome c oxidase. Another putative denitrification gene product shows structural relation to the subunit III of the oxidase. N2O reductase and NO reductase may be ancestors of energy-conserving enzymes of the heme-copper oxidase superfamily. More than 30 genes for denitrification are located in a > 30-kb cluster in Pseudomonas stutzeri, and comparable gene clusters have been identified in Pseudomonas aeruginosa and Paracoccus denitrificans. Genes necessary for nitrite reduction and NO reduction have a mosaic arrangement with very few conserved locations within these clusters and relative to each other.

Two enzymes with a common function but different heme ligands in the forms as isolated. Optical and magnetic properties of the heme groups in the oxidized forms of nitrite reductase, cytochrome cd1, from Pseudomonas stutzeri and Thiosphaera pantotropha.

It is shown that, in the oxidized state, heme c of Pseudomonas stutzeri (ZoBell strain) cytochrome cd1 has histidine-methionine ligation as observed for cytochrome cd1 from Pseudomonas aeruginosa [Sutherland, J., Greenwood, C., Peterson, J., and Thomson, A. J. (1986) Biochem. J. 233, 893-898]. However, the X-ray structure of Thiosphaera pantotropha cytochrome cd1 reveals bis-histidine ligation for heme c. It is confirmed by EPR and near-infrared (NIR) MCD measurements that the bis-histidine coordination remains unaltered in the solution phase. Hence, the difference between the heme c ligation states defines two distinct classes of oxidized cytochromes cd1 as isolated. A weak feature in the T. pantotropha NIR MCD at 1900 nm suggests that a small population of heme c has histidine-methionine coordination. The ligation state of heme d1 cannot be defined with the same level of confidence, because the porphyrin-to-Fe(III) charge-transfer (CT) bands are less well characterized for this class of partially reduced porphyrin ring. However, variable temperature absorption and MCD spectra show that, in the T. pantotropha enzyme, heme d1 exists in a thermal low-spin/high-spin mixture with the low-spin as the ground state, whereas in P. stutzeri cytochrome cd1, and d1 heme is low-spin at all temperatures. A weak band, assigned as the heme d1 porphyrin-pi(a1u,a2u)-to-ferric(d) charge-transfer transition has been identified for the first time at 2170 nm. Its magnetic properties show the heme d1 to have an unusual (dxz,yz)4(dxy)1 electronic ground state as is found for low-spin Fe(III) chlorins [Cheesman, M. R., and Walker, F. A. (1996) J. Am. Chem. Soc. 118, 7373-7380]. It is proposed that the localization of the Fe(III) unpaired d-electron in an orbital lying in the heme plane may decrease the affinity of the Fe(III) heme for unsaturated ligands such as NO. Although heme d1 in the enzymes from P. stutzeri and T. pantotropha shows different temperature-dependent spin properties, the positions of the low-spin Fe(III) alpha-absorption band, at approximately 640 nm, are very similar to those observed for cytochromes cd1 from eight other sources, suggesting that all have similar strength fields from the axial ligands and, hence, that all have the same coordination, namely histidine-tyrosine or possibly histidine-hydroxide at the heme.

Sequence analysis of an internal 9.72-kb segment from the 30-kb denitrification gene cluster of Pseudomonas stutzeri.

The DNA segment was sequenced that links the nir-nor and nos gene clusters for denitrification of Pseudomonas stutzeri ATCC 14405. Of 10 predicted gene products, four are putative membrane proteins. Sequence similarity was detected with the subunit III of cytochrome-c oxidase (ORF175), PQQ3 of the biosynthetic pathway for pyrrolo-quinoline quinone (ORF393), S-adenosylmethionine-dependent uroporphyrinogen-III C-methyltransferase (ORF278), the cytochrome cd1 nitrite reductase and the NirF protein involved in the biosynthesis of heme d1 (ORF507), LysR type transcriptional regulators (ORF286), short-chain alcohol dehydrogenases (ORF247), and a hypothetical protein, YBEC, of Escherichia coli (ORF57). The current data together with previous work establish a contiguous DNA sequence of 29.2 kb comprising the supercluster of nos-nir-nor genes for denitrification in this bacterium.

Mutation of the conserved Cys165 outside of the CuA domain destabilizes nitrous oxide reductase but maintains its catalytic activity. Evidence for disulfide bridges and a putative protein disulfide isomerase gene.

The single conserved Cys165 outside of the CuA domain of nitrous oxide reductase (N2OR) from Pseudomonas stutzeri was mutated to glycine to test its presumed function in metal coordination of the catalytic site, CuZ. The point mutation reduced the cellular level of N2OR 5--10-fold compared to the level of the control strain. In the mutant, the activity and the Cu content of the enzyme, as well as the transcript level of the N2OR structural gene, nosZ, remained unaffected. The mutant enzyme was processed and exported into the periplasm like the wild-type enzyme. Chemical analysis for sulfhydryl groups gave about nine -SH groups/monomer of the apoenzyme prepared from the wild-type enzyme, in accordance with the nine cysteine residues of the derived amino acid sequence. Eight -SH groups were found to form disulfide bridges in the holoenzyme dimer. We propose that in the native state of the enzyme Cys165 does not bind to CuZ, but may be part of a disulfide bridge essential for the stability of N2OR. Immediately downstream of the genes nosDFY, encoding the components for Cu incorporation into the reductase, we have identified the open reading frame, ORFL, whose derived product has the signature of a protein disulfide isomerase.

Characterization of the membranous denitrification enzymes nitrite reductase (cytochrome cd1) and copper-containing nitrous oxide reductase from Thiobacillus denitrificans.

Cytochrome cd1-nitrite reductase and nitrous oxide reductase of Thiobacillus denitrificans were purified and characterized by biochemical and immunochemical methods. In contrast to the generally soluble nature of the denitrification enzymes, these two enzymes were isolated from the membrane fraction of T. denitrificans and remained active after solubilization with Triton X-100. The properties of the membrane-derived enzymes were similar to those of their soluble counterparts from the same organism. Nitrous oxide reductase activity was inhibited by acetylene. Nitrite reductase and nitrous oxide reductase cross-reacted with antisera raised against the soluble enzymes from Pseudomonas stutzeri. The nirS, norBC, and nosZ genes encoding the cytochrome cd1-nitrite reductase, nitric oxide reductase, and nitrous oxide reductase, respectively, from P. stutzeri hybridized with genomic DNA from T. denitrificans. Cross-reactivity and similar N-terminal amino acid and gene sequences suggest that the primary structures of the Thiobacillus enzymes are homologous to the soluble proteins from P. stutzeri.