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.

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.

Discrimination of ascorbate-dependent nonenzymatic and enzymatic, membrane-bound reduction of nitric oxide in denitrifying Pseudomonas perfectomarinus.

The marine nitrite-respiring (denitrifying) bacterium, Pseudomonas perfectomarinus, catalyzes by a membrane-bound enzyme the reduction of nitric oxide to nitrous oxide with ascorbic-reduced phenazine methosulfate as electron donor. The entire nitric oxide-reducing capability of a cell-free system was membrane bound and this process was studied with respect to pH and substrate dependency. The enzymatic process was perturbed by an identical nonenzymatic reduction by iron(II) ascorbate in neutral to alkaline aqueous solution. 2 mol nitric oxide and 1 mol ascorbate were consumed per mol nitrous oxide formed. Enzymatic and nonenzymatic processes were discriminated by their differential behavior towards pH and metal-chelating agents. The pH optimum for the enzymatic and nonenzymatic reaction was 5.2 and greater than 7.0, respectively. EDTA (10 mM) inhibited the nonenzymatic reduction completely without interfering with the membrane-bound activity. The nonenzymatic system mimics the reaction of nitric oxide reductase and could serve as a model to study the formation of the N-N bond in denitrification. Enzymatic generation of nitric oxide by cytochrome cd and subsequent nonenzymatic reduction to nitrous oxide simulate an overall quasi-enzymatic nitrous oxide formation by cytochrome cd. The nonenzymatic reduction of nitric oxide might have occurred in previous work due to the ubiquitous use of ascorbate in studies on nitrite respiration and the likelihood of adventitious iron in biological samples.

Reduction of nitrite to nitrous oxide by a cytoplasmic membrane fraction from the marine denitrifier Pseudomonas perfectomarinus.

A cytoplasmic membrane fraction from the marine denitrifier Pseudomonas perfectomarinus reduced nitrite to nitrous oxide in a stoichiometric reaction without nitric oxide as free intermediate. The membrane system had a specific requirement for FMN with NAD(P)H as electron donors. Other electron donors were ascorbate-reduced cytochrome c-551 or phenazine methosulfate. The membrane fraction contained tightly bound cytochrome cd which represented only a small portion of the total cytochrome cd of the cell. As further terminal oxidase cytochrome o was identified. The membrane fraction produced also nitrous oxide from nitric oxide, however, at a substantially lower rate than from nitrite when using ascorbate-reduced phenazine methosulfate as electron donor.

Multiple nosZ promoters and anaerobic expression of nos genes necessary for Pseudomonas stutzeri nitrous oxide reductase and assembly of its copper centers.

Respiration of N oxides (denitrification) by bacteria is expressed facultatively in response to environmental stimuli. We have studied the transcriptional organization of the nos gene cluster of Pseudomonas stutzeri. This cluster carries the information for a functional nitrous oxide reductase (NosZ) which catalyzes the last step of the denitrification process. The nos genes are transcribed in three units, nosR, nosZ, and nosDFY. Transcription of nosZ is initiated from six different promoters which extend over a region of about 200 bp. The activity of two promoters varies subject to different growth conditions. Promoter P3 is active preferentially under denitrifying conditions and presumably under the control of a homolog of the transcriptional regulator FNR. Promoter P2 is the most active start site under aerobiosis and likely to initiate the low constitutive expression of nosZ. Transcription of nosR, encoding a regulator for nosZ expression, and transcription of the nosDFY operon, required for the copper chromophore assembly of NosZ, are both initiated from a single promoter. Transcription of nosR and the nosDFY operon was shown by phoA and lacZ fusions to be activated under a lowered oxygen tension and the simultaneous presence of an N oxide. The enzymatic activities associated with the hybrid proteins suggest for NosR and NosF a location in the cytoplasmic membrane and the cytoplasm, respectively.

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.

Interdependence of respiratory NO reduction and nitrite reduction revealed by mutagenesis of nirQ, a novel gene in the denitrification gene cluster of Pseudomonas stutzeri.

An open reading frame, designated nirQ, was identified upstream of nirS, the structural gene for the respiratory nitrite reductase of Pseudomonas stutzeri ZoBell. Its derived gene product (275 amino acids, M(r) = 30,554) shows similarity to the NtrC protein family of transcriptional activators. Deletion-replacement mutagenesis of the nirQ gene resulted in the simultaneous loss of nitrite reduction and NO reduction in vivo. However, both reductases were still synthesized, with only nitrite reductase being active in vitro. NO reductase was overproduced by a factor of about 2. Our results indicate that the systems for nitrite reduction and NO reduction are functionally coupled.

The nirSTBM region coding for cytochrome cd1-dependent nitrite respiration of Pseudomonas stutzeri consists of a cluster of mono-, di-, and tetraheme proteins.

Genes for respiratory nitrite reduction (denitrification) of Pseudomonas stutzeri are clustered within 7 kbp. A 4.6-kbp Hind III-Kpn I fragment carrying nirS, the structural gene for cytochrome cd1, was sequenced. An open reading frame immediately downstream of nirS codes for a 22.8-kDa protein with four heme c-binding motifs. Mutagenesis of this gene causes an apparent defect in electron donation to cytochrome cd1. Following this ORF are the structural genes for cytochrome c552, cytochrome c551, and ORF5 that codes for a 11.9-kDa monoheme protein. All cytochromes have a signal sequence for protein export.

The cupric site in nitrous oxide reductase contains a mixed-valence [Cu(II),Cu(I)] binuclear center: a multifrequency electron paramagnetic resonance investigation.

Multifrequency electron paramagnetic resonance (EPR) spectra of the Cu(II) site in nitrous oxide reductase (N2OR) from Pseudomonas stutzeri confirm the assignment of the low field g value at 2.18 consistent with the seven line pattern observed at 9.31 GHz, 10 K. S-band spectra at 20 K are better resolved than the X-band spectra recorded at 10 K. The features observed at 2.4, 3.4, 9.31 and 35 GHz are explained by a mixed-valence [Cu(1.5)..Cu(1.5)] S = 1/2 species with the unpaired electron delocalized between two equivalent Cu nuclei. The resemblance of the N2OR S-band spectra to the spectra for the EPR-detectable Cu of cytochrome c oxidase suggests that the S-band spectrum for cytochrome c oxidase measured below 30 K may also contain hyperfine splittings from two approximately equivalent Cu nuclei.

Structural homologies between the amino acid sequence of Clostridium pasteurianum MoFe protein and the DNA sequences of nifD and K genes of phylogenetically diverse bacteria.

The complete amino acid sequence of the larger (alpha-) subunit and about 70% of the total sequence of the smaller (beta-) subunit of the MoFe protein from Clostridium pasteurianum was determined by analyses of peptides derived from BrCN cleavage and by digestions with trypsin, staphylococcal protease and lysylendo-peptidase of the separated subunits. The alpha-subunit has 529 amino acid residues, giving an Mr value of 58 774. This is the first complete sequence for the alpha-subunit of an isolated MoFe protein. In comparing the sequences of both subunits to those from other sources, 5 out of 9 cysteines in the alpha-subunit and 3 out of 6 in the beta-subunit are invariant, thus suggesting a function as ligands to FeS and MoFeS clusters in the MoFe protein. All of these cysteines are located in the amino terminal halves of both subunits.

A model of the copper centres of nitrous oxide reductase (Pseudomonas stutzeri). Evidence from optical, EPR and MCD spectroscopy.

Nitrous oxide reductase (N2OR), Pseudomonas stutzeri, catalyses the 2 electron reduction of nitrous oxide to di-nitrogen. The enzyme has 2 identical subunits (Mr approximately 70,000) of known amino acid sequence and contains approximately 4 Cu ions per subunit. By measurement of the optical absorption, electron paramagnetic resonance (EPR) and low-temperature magnetic circular dichroism (MCD) spectra of the oxidised state, a semi-reduced form and the fully reduced state of the enzyme it is shown that the enzyme contains 2 distinct copper centres of which one is assigned to an electron-transfer function, centre A, and the other to a catalytic site, centre Z. The latter is a binuclear copper centre with at least 1 cysteine ligand and cycles between oxidation levels Cu(II)/Cu(II) and Cu(II)/Cu(I) in the absence of substrate or inhibitors. The state Cu(II)/Cu(I) is enzymatically inactive. The MCD spectra provide evidence for a second form of centre Z, which may be enzymatically active, in the oxidised state of the enzyme. Centre A is structurally similar to that of CuA in bovine and bacterial cytochrome c oxidase and also contains copper ligated by cysteine. This centre may also be a binuclear copper complex.

Multifrequency EPR evidence for a bimetallic center at the CuA site in cytochrome c oxidase.

Multifrequency electron paramagnetic resonance (EPR) spectra of the Cu(II) site in bovine heart cytochrome c oxidase (COX) and nitrous oxide reductase (N2OR) from Pseudomonas stutzeri confirm the existence of Cu-Cu interaction in both enzymes. C-band (4.5 GHz) proves to be a particularly good frequency complementing the spectra of COX and N2OR recorded at 2.4 and 3.5 GHz. Both the high and low field region of the EPR spectra show the presence of a well-resolved 7-line pattern consistent with the idea of a binuclear Cu center in COX and N2OR. Based on this assumption consistent g-values are calculated for gz and gx at four frequencies. No consistent g-values are obtained with the assumption of a 4-line pattern indicative for a mononuclear Cu site.

Pseudomonas stutzeri ferredoxin: close similarity to Azotobacter vinelandii and Pseudomonas ovalis ferredoxins.

The complete primary structure of Pseudomonas stutzeri strain ZoBell ferredoxin was determined by a combination of protease digestion, Edman degradation, and carboxypeptidase digestion and was: TFVVTDNCIKCKYTDCVEVCPVDCFYEGPNFLVIH PDECIDCALCEPECPAQAIFSEDEVPEDQQEFIELNADLAEVWPNITE KKDALADAEEWDGVKDKLQYLER. The calculated molecular weight was 12,110 excluding iron and sulfur atoms. The amino acid sequence was highly homologous to those of Azotobacter vinelandii and Pseudomonas ovalis ferredoxins. It showed, like the other two, a Tyr-Thr insertion between the second and third Cys, and extra Cys at position 24 and, compared to Clostridium- and Bacillus-type ferredoxins, an extended C-terminal sequence.

Correspondence of the larger subunit of the MoFe-protein in clostridial nitrogenase to the nif D gene products of other N2-fixing organisms.

The amino(N)-terminal sequence of the larger subunit (alpha) of the MoFe-protein from Clostridium pasteurianum was determined up to 179 amino acid residues by analyses of BrCN and tryptic peptides of the original subunit. Apparent similarities exist among the sequence of the clostridial alpha-subunit, that of the smaller subunit (beta) of the Azotobacter vinelandii MoFe protein, and those predicted from the nucleotide sequences of nif D genes in Klebsiella pneumoniae and Anabaena 7120. In comparing the sequences of C. pasteurianum and K. pneumoniae, 45% of residues are identical of a total of 184 sites. Therefore, the larger subunit of the clostridial MoFe-protein must correspond to the nif D gene product of K. pneumoniae.

Ferredoxins from the photosynthetic purple non-sulfur bacterium Rhodopseudomonas palustris. Isolation and amino acid sequence of ferredoxin I.

Two ferredoxins, ferredoxins I and II, were prepared from Rhodopseudomonas palustris. They were separated on a Sephadex column after carboxymethylation and ferredoxin I, the major component, was subjected to an amino acid sequence study. The protein was composed of 63 amino acid residues and the sequence was as follows: (sequence; see text). The molecular weight was calculated to be 6,718, excluding iron and sulfur atoms. The distribution of the nine cysteine residues was similar to but clearly distinct from those of ferredoxins of other photosynthetic bacteria. Comparison of this ferredoxin with those of other bacteria suggests that the photosynthetic bacteria evolved on separate lines. Ferredoxin II was also subjected to analyses of amino acid composition and terminal sequences, but no further study was possible due to the limited material. Although the composition was different from that of ferredoxin I, the terminal sequences were exactly the same as those of ferredoxin I.

The heme-copper oxidase family consists of three distinct types of terminal oxidases and is related to nitric oxide reductase.

Among aerobic prokaryotes, many different terminal oxidase complexes have been described. Sequence comparison has revealed that the aa3-type cytochrome c oxidase and the bo3-type quinol oxidase are variations on the same theme: the heme-copper oxidase. A third member of this family has recently been recognized: the cbb3-type cytochrome c oxidase. Here we give an overview, and report that nitric oxide (NO) reductase, a bc-type cytochrome involved in denitrification, shares important features with these terminal oxidases as well. Tentative structural, functional and evolutionary implications are discussed.