Need for cytochrome bc1 complex for dissimilatory nitrite reduction of Pseudomonas aeruginosa

Pseudomonas aeruginosa strains deficient in the genes for cytochrome c1, a subunit of the cytochrome bc1 complex, or the tetraheme membrane protein NapC, which is similar to NirT of Pseudomonas stutzeri, were constructed and their growth was investigated. The cytochrome c1 mutant could not grow under anaerobic conditions with nitrite as an electron acceptor and did not reduce nitrite in spite of its producing active nitrite reductase. NirM (cytochrome c551) and azurin, which are the direct electron donors for nitrite reductase, were reduced by succinate in the presence of the membrane fraction from the wild-type strain as a mediator but not in the presence of that from the cytochrome c1 mutant. These results indicated that cytochrome bc1 complex was necessary for electron transfer from the membrane quinone pool to nitrite reductase. The NapC mutant grew anaerobically at the expense of nitrite, indicating that NapC was not necessary for nitrite reduction.     

The structural gene for cytochrome c551 from Pseudomonas aeruginosa. The nucleotide sequence shows a location downstream of the nitrite reductase gene

The gene coding for Pseudomonas aeruginosa cytochrome c551 has been cloned and its nucleotide sequence determined. Cytochrome c551 is expressed as a 104 amino acid pre-protein from which a signal peptide of 22 amino acids is cleaved off during the translocation across the cytoplasmic membrane. The gene is located just downstream of the gene coding for nitrite reductase on the Pseudomonas aeruginosa chromosome, suggesting that these genes form an operon.     

The roles of the polytopic membrane proteins NarK, NarU and NirC in Escherichia coli K-12: two nitrate and three nitrite transporters

Two polytopic membrane proteins, NarK and NarU, are assumed to transport nitrite out of the Escherichia coli cytoplasm, but how nitrate enters enteric bacteria is unknown. We report the construction and use of four isogenic strains that lack nitrate reductase Z and the periplasmic nitrate reductase, but express all combinations of narK and narU. The active site of the only functional nitrate reductase, nitrate reductase A, is located in the cytoplasm, so nitrate reduction by these four strains is totally dependent upon a mechanism for importing nitrate. These strains were exploited to determine the roles of NarK and NarU in both nitrate and nitrite transport. Single mutants that lack either NarK or NarU were competent for nitrate-dependent anaerobic growth on a non-fermentable carbon source, glycerol. They transported and reduced nitrate almost as rapidly as the parental strain. In contrast, the narK-narU double mutant was defective in nitrate-dependent growth unless nitrate transport was facilitated by the nitrate ionophore, reduced benzyl viologen (BV). It was also unable to catalyse nitrate reduction in the presence of physiological electron donors. Synthesis of active nitrate reductase A and the cytoplasmic, NADH-dependent nitrite reductase were unaffected by the narK and narU mutations. The rate of nitrite reduction catalysed by the cytoplasmic, NADH-dependent nitrite reductase by the double mutant was almost as rapid as that of the NarK+-NarU+ strain, indicating that there is a mechanism for nitrite uptake by E. coli that is in-dependent of either NarK or NarU. The nir operon encodes a soluble, cytoplasmic nitrite reductase that catalyses NADH-dependent reduction of nitrite to ammonia. One additional component that contributes to nitrite uptake was shown to be NirC, the hydrophobic product of the third gene of the nir operon, which is predicted to be a polytopic membrane protein with six membrane-spanning helices. Deletion of both NarK and NirC decreased nitrite uptake and reduction to a basal rate that was fully restored by a single chromosomal copy of either narK or nirC. A multicopy plasmid encoding NarU complemented a narK mutation for nitrite excretion, but not for nitrite uptake. We conclude that, in contrast to NirC, which transports only nitrite, NarK and NarU provide alternative mechanisms for both nitrate and nitrite transport. However, NarU might selectively promote nitrite ex-cretion, not nitrite uptake.     

The role of blood platelets in tumor angiogenesis

The purple photosynthetic bacterium Rubrivivax gelatinosus has, at least, four periplasmic electron carriers, i.e., HiPIP, two cytochromes c?with low- and high-midpoint potentials, and cytochrome c? as electron donors to the photochemical reaction center. The quadruple mutant lacking all four electron carrier proteins showed extremely slow photosynthetic growth. During the long-term cultivation of this mutant under photosynthetic conditions, a suppressor strain recovering the wild-type growth level appeared. In the cells of the suppressor strain, we found significant accumulation of a soluble c-type cytochrome that has not been detected in wild-type cells. This cytochrome c has a redox midpoint potential of about +280 mV and could function as an electron donor to the photochemical reaction center in vitro. The amino acid sequence of this cytochrome c was 65% identical to that of the high-potential cytochrome c?of this bacterium. The gene for this cytochrome c was identified as nirM on the basis of its location in the newly identified nir operon, which includes a gene coding cytochrome cd?-type nitrite reductase. Phylogenetic analysis and the well-conserved nir operon gene arrangement suggest that the origin of the three cytochromes c? in this bacterium is NirM. The two other cytochromes c?, of high and low potentials, proposed to be generated by gene duplication from NirM, have evolved to function in distinct pathways.     

A NapC/NirT-type cytochrome c (NrfH) is the mediator between the quinone pool and the cytochrome c nitrite reductase of Wolinella succinogenes

Wolinella succinogenes can grow by anaerobic respiration with nitrate or nitrite using formate as electron donor. Two forms of nitrite reductase were isolated from the membrane fraction of W. succinogenes. One form consisted of a 58 kDa polypeptide (NrfA) that was identical to the periplasmic nitrite reductase. The other form consisted of NrfA and a 22 kDa polypeptide (NrfH). Both forms catalysed nitrite reduction by reduced benzyl viologen, but only the dimeric form catalysed nitrite reduction by dimethylnaphthoquinol. Liposomes containing heterodimeric nitrite reductase, formate dehydrogenase and menaquinone catalysed the electron transport from formate to nitrite; this was coupled to the generation of an electrochemical proton potential (positive outside) across the liposomal membrane. It is concluded that the electron transfer from menaquinol to the catalytic subunit (NrfA) of W. succinogenes nitrite reductase is mediated by NrfH. The structural genes nrfA and nrfH were identified in an apparent operon (nrfHAIJ) with two additional genes. The gene nrfA encodes the precursor of NrfA carrying an N-terminal signal peptide (22 residues). NrfA (485 residues) is predicted to be a hydrophilic protein that is similar to the NrfA proteins of Sulfurospirillum deleyianum and of Escherichia coli. NrfH (177 residues) is predicted to be a membrane-bound tetrahaem cytochrome c belonging to the NapC/NirT family. The products of nrfI and nrfJ resemble proteins involved in cytochrome c biogenesis. The C-terminal third of NrfI (902 amino acid residues) is similar to CcsA proteins from Gram-positive bacteria, cyanobacteria and chloroplasts. The residual N-terminal part of NrfI resembles Ccs1 proteins. The deduced NrfJ protein resembles the thioredoxin-like proteins (ResA) of Helicobacter pylori and of Bacillus subtilis, but lacks the common motif CxxC of ResA. The properties of three deletion mutants of W. succinogenes (DeltanrfJ, DeltanrfIJ and DeltanrfAIJ) were studied. Mutants DeltanrfAIJ and DeltanrfIJ did not grow with nitrite as terminal electron acceptor or with nitrate in the absence of NH4+ and lacked nitrite reductase activity, whereas mutant DeltanrfJ showed wild-type properties. The NrfA protein formed by mutant DeltanrfIJ seemed to lack part of the haem C, suggesting that NrfI is involved in NrfA maturation.     

Crystal Structure of Dihydro-Heme d1 Dehydrogenase NirN from Pseudomonas aeruginosa Reveals Amino Acid Residues Essential for Catalysis

Many bacteria can switch from oxygen to nitrogen oxides, such as nitrate or nitrite, as terminal electron acceptors in their respiratory chain. This process is called “denitrification” and enables biofilm formation of the opportunistic human pathogen Pseudomonas aeruginosa, making it more resilient to antibiotics and highly adaptable to different habitats. The reduction of nitrite to nitric oxide is a crucial step during denitrification. It is catalyzed by the homodimeric cytochrome cd₁ nitrite reductase (NirS), which utilizes the unique isobacteriochlorin heme d₁ as its reaction center. Although the reaction mechanism of nitrite reduction is well understood, far less is known about the biosynthesis of heme d₁. The last step of its biosynthesis introduces a double bond in a propionate group of the tetrapyrrole to form an acrylate group. This conversion is catalyzed by the dehydrogenase NirN via a unique reaction mechanism. To get a more detailed insight into this reaction, the crystal structures of NirN with and without bound substrate have been determined. Similar to the homodimeric NirS, the monomeric NirN consists of an eight-bladed heme d₁-binding β-propeller and a cytochrome c domain, but their relative orientation differs with respect to NirS. His147 coordinates heme d₁ at the proximal side, whereas His323, which belongs to a flexible loop, binds at the distal position. Tyr461 and His417 are located next to the hydrogen atoms removed during dehydrogenation, suggesting an important role in catalysis. Activity assays with NirN variants revealed the essentiality of His147, His323 and Tyr461, but not of His417.     

Pseudoazurin mediates periplasmic electron flow in a mutant strain of Paracoccus denitrificans lacking cytochrome c550

A periplasmic protein able to transfer electrons from cytoplasmic membrane to the periplasmic nitrite reductase (cytochrome cd1) has been purified from the anoxically grown cytochrome c550 mutant strain Pd2121 and shown to be pseudoazurin by several independent criteria (molecular mass, copper content, visible spectrum, N-terminal amino acid sequence). Under our assay conditions, the half-saturation of electron transport occurred at about 10 microM pseudoazurin; the reaction was retarded by increasing ionic strength.     

Controlled digestion with trypsin as a structural probe for the N-terminal peptide of soluble and membranous cytochrome c.

When purified bovine cytochrome c1 is digested with trypsin under controlled conditions, the heme polypeptide is preferentially converted from a species of molecular weight 30,600 to a heme polypeptide of molecular weight 29,000. The trypsin sensitive peptide bond is located in the N-terminal region of the cytochrome. Both the reduced and oxidized cytochrome are susceptible to hydrolysis by trypsin at the same locus, but the reduced cytochrome is cleaved at an initial rate approximately twofold greater than the oxidized cytochrome. Membranous cytochrome c1, as occurring in cytochrome b-c1 complex or succinate-cytochrome c reductase complex, is not susceptible to trypsin proteolysis under similar conditions, nor after more extensive treatment of the membranes with trypsin, in spite of the fact that cytochrome c1 presumably comes into contact with cytochrome c at the membrane surface during electron transport. These findings are consistent with a model for the structure of cytochrome c1 in situ in which the cytochrome is an integral membrane protein, located primarily in the membrane continuum, while still having the heme-containing portion of the protein available at the membrane surface for electron transfer to cytochrome c.     

A single channel for nitrate uptake, nitrite export and nitrite uptake by Escherichia coli NarU and a role for NirC in nitrite export and uptake

Two related polytopic membrane proteins of the major facilitator family, NarK and NarU, catalyse nitrate uptake, nitrite export and nitrite uptake across the Escherichia coli cytoplasmic membrane by an unknown mechanism. A 12-helix model of NarU was constructed based upon six alkaline phosphatase and beta-galactosidase fusions to NarK and the predicted hydropathy for the NarK family. Fifteen residues conserved in the NarK-NarU protein family were substituted by site-directed mutagenesis, including four residues that are essential for nitrate uptake by Aspergillus nidulans: arginines Arg(87) and Arg(303) in helices 2 and 8, and two glycines in a nitrate signature motif. Despite the wide range of substitutions studied, in no case did mutation result in loss of one biochemical function without simultaneous loss of all other functions. A NarU+ NirC+ strain grew more rapidly and accumulated nitrite more rapidly than the isogenic NarU+ NirC(-) strain. Only the NirC+ strain consumed nitrite rapidly during the later stages of growth. Under conditions in which the rate of nitrite reduction was limited by the rate of nitrite uptake, NirC+ strains reduced nitrite up to 10 times more rapidly than isogenic NarU+ strains, indicating that both nitrite efflux and nitrite uptake are largely dependent on NirC. Isotope tracer experiments with [15N]nitrate and [14N]nitrite revealed that [15N]nitrite accumulated in the extracellular medium even when there was a net rate of nitrite uptake and reduction. We propose that NarU functions as a single channel for nitrate uptake and nitrite expulsion, either as a nitrate-nitrite antiporter, or more likely as a nitrate/H+ or nitrite/H+ channel.     

Anaerobic activation of the entire denitrification pathway in Pseudomonas aeruginosa requires Anr, an analog of Fnr.

The Pseudomonas aeruginosa gene anr, which encodes a structural and functional analog of the anaerobic regulator Fnr in Escherichia coli, was mapped to the SpeI fragment R, which is at about 59 min on the genomic map of P. aeruginosa PAO1. Wild-type P. aeruginosa PAO1 grew under anaerobic conditions with nitrate, nitrite, and nitrous oxide as alternative electron acceptors. An anr deletion mutant, PAO6261, was constructed. It was unable to grow with these alternative electron acceptors; however, its ability to denitrify was restored upon the introduction of the wild-type anr gene. In addition, the activities of two enzymes in the denitrification pathway, nitrite reductase and nitric oxide reductase, were not detectable under oxygen-limiting conditions in strain PAO6261 but were restored when complemented with the anr+ gene. These results indicate that the anr gene product plays a key role in anaerobically activating the entire denitrification pathway.     

Biodiversity of the P450 catalytic cycle: yeast cytochrome b5/NADH cytochrome b5 reductase complex efficiently drives the entire sterol 14-demethylation (CYP51) reaction

The widely accepted catalytic cycle of cytochromes P450 (CYP) involves the electron transfer from NADPH cytochrome P450 reductase (CPR), with a potential for second electron donation from the microsomal cytochrome b5/NADH cytochrome b5 reductase system. The latter system only supported CYP reactions inefficiently. Using purified proteins including Candida albicans CYP51 and yeast NADPH cytochrome P450 reductase, cytochrome b5 and NADH cytochrome b5 reductase, we show here that fungal CYP51 mediated sterol 14alpha-demethylation can be wholly and efficiently supported by the cytochrome b5/NADH cytochrome b5 reductase electron transport system. This alternative catalytic cycle, where both the first and second electrons were donated via the NADH cytochrome b5 electron transport system, can account for the continued ergosterol production seen in yeast strains containing a disruption of the gene encoding CPR.     

Oxidation by nitrite of azurin and cytochrome c-551 from Pseudomonas aeruginosa

The nitrite oxidizes reduced azurin and cytochrome c-551 from Pseudomonas aeruginosa. The effects of pH, ionic strength and concentrations of nitrite, EDTA and the protein on the oxidation were investigated. The results obtained indicate that nitrite interacts not only with the terminal electron carrier of the nitrite reducing chain (nitrite reductase, cytochrome cd1) but also with the intermediate electron carrier components of the chain (azurin and cytochrome c-551).     

Change in the ratio of cytochrome oxidase activity to nitrite reductase activity of Pseudomonas aeruginosa nitrite reductase with the kind of C-type cytochrome used as an electron donor.

The ratio between the nitrite reductase and cytochrome oxidase activities of Pseudomonas aeruginosa nitrite reductase [EC 1.9.3.2.] varies with kind of C-type cytochrome used as the electron donor. Withe cytochrome c-548, 554 (Micrococcus sp.), the nitrite reductase activity is greater than the cytochrome oxidase activity, while the former is smaller than the latter with cytochrome c-554 (Navicula pelliculosa). The aerobic oxidation catalyzed by this enzyme of denitrifying bacterial ferrocytochrome c is greatly accelerated on addition of nitrite, while that of the algal ferrocytochrome c is not affected or is even depressed by the salt. An accelerative effect of nitrite is generally observed with many kinds of C-type cytochromes which react with the enzyme very or fairly rapidly. The difference in the ratio of the two activities of the enzyme seems to arise according to whether or not nitrite affects the interaction of C-type cytochrome with the enzyme.     

A cytochrome cd 1-type nitrite reductase mediates the first step of denitrification in Alcaligenes eutrophus

Respiratory nitrite reductase (NIR) has been purified from the soluble extract of denitrifying cells of Alcaligenes eutrophus strain H16 to apparent electrophoretic homogeneity. The enzyme was induced under anoxic conditions in the presence of nitrite. Purified NIR showed typical features of a cytochrome cd ₁-type nitrite reductase. It appeared to be a dimer of 60 kDa subunits, its activity was only weakly inhibited by the copper chelator diethyldithiocarbamate, and spectral analysis revealed absorption maxima which were characteristic for the presence of heme c and heme d ₁. The isoelectric point of 8.6 was considerably higher than the pI determined for cd ₁ nitrite reductases from pseudomonads. Eighteen amino acids at the N-terminus of the A. eutrophus NIR, obtained by protein sequencing, showed no significant homology to the N-terminal region of nitrite reductases from Pseudomonas stutzeri and Pseudomonas aeruginosa.     

Domain swing upon His to Ala mutation in nitrite reductase of Pseudomonas aeruginosa

The nitrite reductase (NIR) from Pseudomonas aeruginosa (NIR-Pa) is a soluble enzyme catalysing the reduction of nitrite (NO2(-)) to nitric oxide (NO). The enzyme is a 120 kDa homodimer, in which the monomers carry a c-heme domain and a d(1)-heme domain. The structures of the enzyme in both the oxidised and reduced state were solved previously and indicate His327 and His369 as putative catalytic residues. The kinetic characterisation of site-directed mutants has shown that the substitution of either one of these two His with Ala dramatically reduces the physiologically relevant reactivity towards nitrite, leaving the reactivity towards oxygen unaffected. The three-dimensional structures of P. aeruginosa NIR mutant H327A, and H369A in complex with NO have been solved by multiple wavelength anomalous dispersion (MAD), using the iron anomalous signal, and molecular replacement techniques. In both refined crystal structures the c-heme domain, whilst preserving its classical c-type cytochrome fold, has undergone a 60 degrees rigid-body rotation around an axis parallel with the pseudo 8-fold axis of the beta-propeller, and passing through residue Gln115. Even though the distance between the Fe ions of the c and d(1)-heme remains 21 A, the edge-to-edge distance between the two hemes has increased by 5 A. Furthermore the distal side of the d(1)-heme pocket appears to have undergone structural re-arrangement and Tyr10 has moved out of the active site. In the H369A-NO complex, the position and orientation of NO is significantly different from that of the NO bound to the reduced wild-type structure. Our results provide insight into the flexibility of the enzyme and the distinction between nitrite and oxidase reduction mechanisms. Moreover they demonstrate that the two histidine residues play a crucial role in the physiological activity of nitrite reduction, ligand binding and in the structural organisation of nitrite reductase from P. aeruginosa.     

NirN Protein from Pseudomonas aeruginosa is a Novel Electron-bifurcating Dehydrogenase Catalyzing the Last Step of Heme d1 Biosynthesis

Heme d₁ plays an important role in denitrification as the essential cofactor of the cytochrome cd₁ nitrite reductase NirS. At present, the biosynthesis of heme d₁ is only partially understood. The last step of heme d₁ biosynthesis requires a so far unknown enzyme that catalyzes the introduction of a double bond into one of the propionate side chains of the tetrapyrrole yielding the corresponding acrylate side chain. In this study, we show that a Pseudomonas aeruginosa PAO1 strain lacking the NirN protein does not produce heme d₁. Instead, the NirS purified from this strain contains the heme d₁ precursor dihydro-heme d₁ lacking the acrylic double bond, as indicated by UV-visible absorption spectroscopy and resonance Raman spectroscopy. Furthermore, the dihydro-heme d₁ was extracted from purified NirS and characterized by UV-visible absorption spectroscopy and finally identified by high-resolution electrospray ionization mass spectrometry. Moreover, we show that purified NirN from P. aeruginosa binds the dihydro-heme d₁ and catalyzes the introduction of the acrylic double bond in vitro. Strikingly, NirN uses an electron bifurcation mechanism for the two-electron oxidation reaction, during which one electron ends up on its heme c cofactor and the second electron reduces the substrate/product from the ferric to the ferrous state. On the basis of our results, we propose novel roles for the proteins NirN and NirF during the biosynthesis of heme d₁.     

A cytochrome cd1-type nitrite reductase isolated from the marine denitrifier Pseudomonas nautica 617: purification and characterization

Nitrite reductase (cytochrome cd1) was purified to electrophoretic homogeneity from the soluble extract of the marine denitrifying bacterium Pseudomonas nautica strain 617. Cells were anaerobically grown with 10 mM nitrate as final electron acceptor. The soluble fraction was purified by four successive chromatographic steps and the purest cytochrome cd1 exhibited an A280 nm(oxidized)/A410nm(oxidized) coefficient of 0.90. In the course of purification, cytochrome cd1 specific activity presented a maximum value of 0.048 units/mg of protein. This periplasmic enzyme is a homodimer and each 60 kDa subunit contains one heme c and one heme d1 as prosthetic moieties, both in a low spin state. Redox potentials of hemes c and d1 were determined at three different pH values (6.6, 7.6 and 8.6) and did not show any pH dependence. The first 20 amino acids of the NH2-terminal region of the protein were identified and the sequence showed 45% identity with the corresponding region of Pseudomonas aeruginosa nitrite reductase but no homology to Pseudomonas stutzeri and Paracoccus denitrificans enzymes. Spectroscopic properties of Pseudomonas nautica 617 cytochrome cd1 in the ultraviolet-visible range and in electron paramagnetic resonance are described. The formation of a heme d1 -nitric-oxide complex as an intermediate of nitrite reduction was demonstrated by electron paramagnetic resonance experiments.     

Laboratoire de Chimie Bactérienne C.N.R.S., Marsielle, France.

Summary Mutants of E. coli, completely devoid of nitrite reductase activity with glucose or formate as donor were studied. Biochemical analysis indicates that they are simultaneously affected in nitrate reductase, nitrite reductase, fumarate reductase and hydrogenase activities as well as in cytochrome c₅₅₂ biosynthesis. The use of an antiserum specific for nitrate reductase shows that the nitrate reductase protein is probably missing. A single mutation is responsible for this phenotype: the gene affected, nir R, is located close to tyr R i.e. at 29 min on the chromosomal map.Abbreviations BV Benzyl-Viologen - NTG N-methyl-N-nitro-N-nitrosoguanidine - NR nitrate reductase - NIR nitrite reductase - FR fumarate reductase - HYD hydrogenase - CYT c₅₅₂ cytochrome c₅₅₂     

Nitrite controls the release of nitric oxide in Pseudomonas aeruginosa cd1 nitrite reductase

Nitrite reductase (cd1NIR) from Pseudomonas aeruginosa, which catalyses the reduction of nitrite to nitric oxide (NO), contains a c-heme as the electron acceptor and a d1-heme where catalysis occurs. Reduction involves binding of nitrite to the reduced d1-heme, followed by dehydration to yield NO; release of NO and re-reduction of the enzyme close the cycle. Since NO is a powerful inhibitor of ferrous hemeproteins, enzymatic turnover demands the release of NO. We recently discovered that NO dissociation from the ferrous d1-heme is fast, showing that cd1NIR behaves differently from other hemeproteins. Here we demonstrate for the first time that the physiological substrate nitrite displaces NO from the ferrous enzyme, which enters a new catalytic cycle; this reaction depends on the conserved His369 whose role in substrate stabilization is crucial for catalysis. Thus we suggest that also in vivo the activity of cd1NIR is controlled by nitrite.     

Parallel Pathways for Nitrite Reduction during Anaerobic Growth in Thermus thermophilus

Respiratory reduction of nitrate and nitrite is encoded in Thermus thermophilus by the respective transferable gene clusters. Nitrate is reduced by a heterotetrameric nitrate reductase (Nar) encoded along transporters and regulatory signal transduction systems within the nitrate respiration conjugative element (NCE). The nitrite respiration cluster (nic) encodes homologues of nitrite reductase (Nir) and nitric oxide reductase (Nor). The expression and role of the nirSJM genes in nitrite respiration were analyzed. The three genes are expressed from two promoters, one (nirSp) producing a tricistronic mRNA under aerobic and anaerobic conditions and the other (nirJp) producing a bicistronic mRNA only under conditions of anoxia plus a nitrogen oxide. As for its nitrite reductase homologues, NirS is expressed in the periplasm, has a covalently bound heme c, and conserves the heme d₁ binding pocket. NirJ is a cytoplasmic protein likely required for heme d₁ synthesis and NirS maturation. NirM is a soluble periplasmic homologue of cytochrome c₅₅₂. Mutants defective in nirS show normal anaerobic growth with nitrite and nitrate, supporting the existence of an alternative Nir in the cells. Gene knockout analysis of different candidate genes did not allow us to identify this alternative Nir protein but revealed the requirement for Nar in NirS-dependent and NirS-independent nitrite reduction. As the likely role for Nar in the process is in electron transport through its additional cytochrome c periplasmic subunit (NarC), we concluded all the Nir activity takes place in the periplasm by parallel pathways.