Cytochrome Cd1 Nitrite Reductase Research Articles

Engineering neuroglobin nitrite reductase activity based on myoglobin models

Neuroglobin is a hemoprotein expressed in several nervous system cell lineages with yet unknown physiological functions. Neuroglobin presents a very similar structure to that of the related globins hemoglobin and myoglobin, but shows an hexacoordinate heme as compared to the pentacoordinated heme of myoglobin and hemoglobin. While several reactions of neuroglobin have been characterized in vitro, the relative importance of most of those reactions in vivo is yet undefined. Neuroglobin, like other heme proteins, can reduce nitrite to nitric oxide, providing a possible route to generate nitric oxide in vivo in low oxygen conditions. The reaction kinetics are highly dependent on the nature of the distal residue, and replacement of the distal histidine His64(E7) can increase the reaction rate constants by several orders of magnitude. However, mutation of other distal pocket positions such as Phe28(B10) or Val68(E11) has more limited impact on the rates. Computational analysis using myoglobin as template, guided by the structure of dedicated nitrite reductases like cytochrome cd1 nitrite reductase, has pointed out that combined mutations of the residues B10 and CD1 could increase the nitrite reductase activity of myoglobin, by mimicking the environment of the distal heme pocket in cytochrome cd1 nitrite reductase. As neuroglobin shows high sequence and structural homology with myoglobin, we hypothesized that such mutations (F28H and F42Y in neuroglobin) could also modify the nitrite reductase activity of neuroglobin. Here we study the effect of these mutations. Unfortunately, we do not observe in any case an increase in the nitrite reduction rates. Our results provide some further indications of nitrite reductase regulation in neuroglobin and highlight the minor but critical differences between the structure of penta- and hexacoordinate globins.

Soil anammox and denitrification processes connected with N cycling genes co-supporting or contrasting under different water conditions

The anaerobic ammonium oxidizing bacteria (anammox) are the hidden culprit behind the excessive nitrogen loss under a favorable environment, since their detection and abundance get disturbed by several unknown factors. This study intends to find the gap between actual anammox working capacity under different water conditions and fertilizers in the laboratory. The 15N-isotopic tracer technique was used to measure anammox and denitrification rate, and anammox community structure was analyzed through high-throughput sequencing with cytochrome cd-1 nitrite reductase functional gene (an_nirS gene, initially found in Candidatus Scalindua). The experiment consisted of four treatments, i.e., (I) CK_ Control, (II) UR_Urea, (III) PM_Pig Manure, and (IV) SRF_ Slow release fertilizer, under two water conditions, i.e., (a) Continuous flooding_ CF, (b) Alternate wetting and drying_ AWD. Results showed that anammox under CF decreased over time by −40.24%, and denitrification increased up to 39.25%. However, anammox activity under AWD increased up to 10.62% with the availability of NO2–, and surprisingly accompanied by the reduction in denitrification loss (-31.97%), being the most critical factor. We found that soil nifH and AOB genes were strongly favorable for anammox activity, while we observed the presence of anammox and AOB genes co-existing at the same time in paddy soil. The high-throughput sequencing with an_nirS functional gene showed a much higher diversity of anammox genera ever reported, mostly uncultured and unidentified. We concluded that water management is more prominent than fertilizer for anammox, and the most critical factor is the duration of AWD cycle, because of short term air supply could boost anammox activity and gene abundance, and could reduce denitrifier activity as well as nirK gene abundance.

Synergistic Partial-Denitrification, Anammox, and in-situ Fermentation (SPDAF) Process for Advanced Nitrogen Removal from Domestic and Nitrate-Containing Wastewater.

This study presents a new method for energy-efficient wastewater treatment that synergizes the partial-denitrification, anammox, and in-situ fermentation (SPDAF) processes in an up-flow reactor. Nitrate-containing wastewater and actual domestic sewage were fed into this SPDAF system, which was operated for 180 days without the addition of external carbon sources and aeration. The total inorganic nitrogen (TIN) removal efficiency reached 93.1% with a low C/N ratio of 1.6, a NO3--N/NH4+-N ratio of 1.13 and a TIN concentration of 92.5 mg N/L. The contribution of anammox to nitrogen removal accounted for 95.6%. Batch tests demonstrated that the partial-denitrification process was able to use organics from either the influent or those produced by fermentation, thus providing nitrite for anammox. Significantly, fermentation played a key role in using the slowly biodegradable organics and provided adequate electron donor for partial-denitrification. Metagenomic sequencing analysis showed that the genera related to partial-denitrification, anammox, and fermentation bacteria were coexisted in this SPDAF system. The key functional genes of anammox bacteria (Hzs, 3986 hits; Hdh, 2804 hits) were highly detected in this study. The abundances of cytoplasmic nitrate reductase (58 706 hits) and periplasmic nitrate reductase (70 540 hits) were much higher than copper nitrite reductase (16 436 hits) and cytochrome cd1 nitrite reductase (14 264 hits), potentially contributing to the occurrence of partial-denitrification. Moreover, different abundances of genes involved in fermentation metabolism suggested that fermentation likely generated easily biodegradable organics for partial-denitrification.

Nitrate removal performance and diversity of active denitrifying bacteria in denitrification reactors using poly(L-lactic acid) with enhanced chemical hydrolyzability.

Poly(L-lactic acid) (PLLA) can be used as an external electron donor in denitrification reactors to treat drinking water, aquaculture water, and industrial wastewater with an imbalanced carbon/nitrogen ratio. However, for PLLA to function in these applications, its chemical hydrolyzability requires improvement. Although the adjustment of the crystallinity (Xc) is effective in improving the hydrolyzability of PLLA, the condition for the Xc of PLLA, in which a sufficient amount of lactic acid is released for denitrification, must be clarified. Therefore, this study investigated the effective Xc range and optimal PLLA content as an electron donor for continuous nitrate removal in denitrification reactors. This study also explored the abundance, succession, and diversity of active denitrifying bacteria in denitrification reactors. The nitrate removal activity of activated sludge using the highly crystalline PLLA (Xc = 39.4%) was 1.8 mg NO3- -N g MLSS-1 h-1, which is 2.4 times higher than that using the nearly amorphous PLLA (Xc = 0.9%). During the 57 days of operation, the denitrification reactor with 3% (w/v) highly crystalline PLLA continued to completely remove nitrate, with a maximum nitrate removal activity of 22.8 mg NO3- -N g MLSS-1 h-1. The 16S rRNA amplicon sequencing and clone library analyses are using transcripts of two nitrite reductase genes, encoding cytochrome cd1 nitrite reductase, and copper-containing nitrite reductase revealed that bacteria belonging to the families Comamonadaceae, Rhodocyclaceae, and Alcaligenaceae were active denitrifying bacteria in the denitrification reactor using PLLA.

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.

A three-domain copper-nitrite reductase with a unique sensing loop.

Dissimilatory nitrite reductases are key enzymes in the denitrification pathway, reducing nitrite and leading to the production of gaseous products (NO, N2O and N2). The reaction is catalysed either by a Cu-containing nitrite reductase (NirK) or by a cytochrome cd 1 nitrite reductase (NirS), as the simultaneous presence of the two enzymes has never been detected in the same microorganism. The thermophilic bacterium Thermus scotoductus SA-01 is an exception to this rule, harbouring both genes within a denitrification cluster, which encodes for an atypical NirK. The crystal structure of TsNirK has been determined at 1.63 Å resolution. TsNirK is a homotrimer with subunits of 451 residues that contain three copper atoms each. The N-terminal region possesses a type 2 Cu (T2Cu) and a type 1 Cu (T1CuN) while the C-terminus contains an extra type 1 Cu (T1CuC) bound within a cupredoxin motif. T1CuN shows an unusual Cu atom coordination (His2-Cys-Gln) compared with T1Cu observed in NirKs reported so far (His2-Cys-Met). T1CuC is buried at ∼5 Å from the molecular surface and located ∼14.1 Å away from T1CuN; T1CuN and T2Cu are ∼12.6 Å apart. All these distances are compatible with an electron-transfer process T1CuC → T1CuN → T2Cu. T1CuN and T2Cu are connected by a typical Cys-His bridge and an unexpected sensing loop which harbours a SerCAT residue close to T2Cu, suggesting an alternative nitrite-reduction mechanism in these enzymes. Biophysicochemical and functional features of TsNirK are discussed on the basis of X-ray crystallography, electron paramagnetic resonance, resonance Raman and kinetic experiments.

Syntrophic Growth of Geobacter sulfurreducens Accelerates Anaerobic Denitrification.

Nitrate is considered as a contamination since it’s over discharging to water incurs environmental problems. However, nitrate is an ideal electron sink for anaerobic pollutant degraders desiring electron acceptors due to the high redox potential. Unfortunately, not all degraders can directly reduce nitrate, and the anaerobic direct interspecies electron transfer (DIET) between degraders and denitrifiers has not been confirmed yet. Here we demonstrated that syntrophic growth of Geobacter sulfurreducens PCA with denitrifying microbial community at anaerobic condition eliminated the lag phase of 15 h and improved the denitrification rate by 13∼51% over a broad C/N ratio of 0.5 to 9. Quantitative PCR revealed that G. sulfurreducens selectively enhanced the expression of nirS coding for a cytochrome cd1-nitrite reductase, resulting in a fast and more complete denitrification. Geobacter also selectively enriched its potential denitrifying partners – Diaphorobacter, Delftia, and Shinella – to form spherical aggregates. More studies of the binary culture system need to be carried out to confirm the syntrophic mechanism of Geobacter and denitrifiers in the future. These findings extend our knowledge on understanding the anaerobic bacterial interspecies electron transfer in the denitrification process, which has broader implications in fast selection and stabilization of denitrifiers in wastewater treatment plant, and general understanding of ecology for nitrogen and metal cycling.

Redundant roles of Bradyrhizobium oligotrophicum Cu-type (NirK) and cd1-type (NirS) nitrite reductase genes under denitrifying conditions.

Reduction of nitrite to nitric oxide gas by respiratory nitrite reductases (NiRs) is the key step of denitrification. Denitrifiers are strictly divided into two functional groups based on whether they possess the copper-containing nitrite reductase (CuNiR) encoded by nirK or the cytochrome cd1 nitrite reductase (cdNiR) encoded by nirS. Recently, some organisms carrying both nirK and nirS genes have been found. Bradyrhizobium oligotrophicum S58 is a nitrogen-fixing oligotrophic bacterium that carries a set of genes for complete denitrification of nitrate to dinitrogen, including nirK and nirS genes. We show that denitrification in S58 is functional under low-oxygen conditions (anaerobiosis and microaerobiosis), but not under aerobiosis. Under denitrifying conditions, the ΔnirK and ΔnirS single S58 mutants grew normally and their NiR activity was not affected. However, the ΔnirKS double mutant grew more slowly, presumably because the impaired NiR activity resulted in nitrite accumulation in the medium. These results suggest a redundant role for nirK and nirS genes in B. oligotrophicum S58 denitrification. In addition, we found that the nirS gene product, but not that of nirK, maintains swimming motility of S58 under aerobic and low-oxygen conditions in the presence of nitrate.

Potential application of aerobic denitrifying bacterium Pseudomonas aeruginosa PCN-2 in nitrogen oxides (NOx) removal from flue gas

Conventional biological removal of nitrogen oxides (NOx) from flue gas has been severely restricted by the presence of oxygen. This paper presents an efficient alternative for NOx removal at varying oxygen levels using the newly isolated bacterial strain Pseudomonas aeruginosa PCN-2 which was capable of aerobic and anoxic denitrification. Interestingly, nitric oxide (NO), as the obligatory intermediate, was negligibly accumulated during nitrate and nitrite reduction. Moreover, normal nitrate reduction with decreasing NO accumulation was realized under O2 concentration ranging from 0 to 100%. Reverse transcription and real-time quantitative polymerase chain reaction (RT-qPCR) analysis revealed that high efficient NO removal was attributed to the coordinate regulation of gene expressions including napA (for periplasmic nitrate reductase), nirS (for cytochrome cd1 nitrite reductase) and cnorB (for NO reductase). Further batch experiments demonstrated the immobilized strain PCN-2 possessed high capability of removing NO and nitrogen dioxide (NO2) at O2 concentration of 0–10%. A biotrickling filter established with present strain achieved high NOx removal efficiencies of 91.94–96.74% at inlet NO concentration of 100–500ppm and O2 concentration of 0–10%, which implied promising potential applications in purifying NOx contaminated flue gas.

Electron transfer and docking between cytochrome cd1 nitrite reductase and different redox partners — A comparative study

Cytochrome cd1 nitrite reductases (cd1NiRs) catalyze the reduction of nitrite to nitric oxide in denitrifying bacteria, such as Marinobacter hydrocarbonoclasticus. Previous work demonstrated that the enzymatic activity depends on a structural pre-activation triggered by the entry of electrons through the electron transfer (ET) domain, which houses a heme c center. The catalytic activity of M. hydrocarbonoclasticus cd1NiR (Mhcd1NiR) was tested by mediated electrochemistry, using small ET proteins and chemical redox mediators. The rate of enzymatic reaction depends on the nature of the redox partner, with cytochrome (cyt) c552 providing the highest value. In situations where cyt c552 is replaced by either a biological (cyt c from horse heart) or a chemical mediator the catalytic response was only observed at very low scan rates, suggesting that the intermolecular ET rate is much slower.Molecular docking simulations with the 3D model structure of Mhcd1NiR and cyt c552 or cyt c showed that hydrophobic interactions favor the formation of complexes where the heme c domain of the enzyme is the principal docking site. However, only in the case of cyt c552 the preferential areas of contact and Fe–Fe distances between heme c groups of the redox partners allow establishing competent ET pathways. The coupling of the enzyme with chemical redox mediators was also found not to be energetically favorable. These results indicate that although low activity functional complexes can be formed between Mhcd1NiR and different types of redox mediators, efficient ET is only observed with the putative physiological electron donor cyt c552.

Homeostatic control of nitric oxide (NO) at nanomolar concentrations in denitrifying bacteria - modelling and experimental determination of NO reductase kinetics in vivo in Paracoccus denitrificans.

Homeostatic control of nitric oxide (NO) at nanomolar concentrations appears common among denitrifying bacteria, often ascribed to synchronized expression of nitrite and nitric oxide reductase (Nir and Nor). We questioned whether this is sufficient: using the reported substrate affinities for cytochrome cd1 nitrite reductase (cNor), our model of batch cultures of Paracoccus denitrificans predicted NO concentrations orders of magnitude higher than measured. We rejected a hypothesis that the homeostatic control is due to a negative feedback by NO on the activity of NirS because the inclusion of such feedback resulted in too slow anaerobic growth and N2 production. We proceeded by determining the kinetic parameters for cNor in vivo by a carefully designed experiment, allowing the estimation of NO concentration at the cell surface while anoxic cultures depleted low headspace doses of NO. With the new parameters for cNor kinetics in vivo {v = vmax /[1 + K2 /(NO) + K1 × K2 /(NO)(2) ]; vmax = 3.56 fmol NO cell(-1) h(-1) , K1 < 1 nM, and K2 = 34 nM}, the model predicted NO concentrations close to that measured. Thus, enzyme kinetics alone can explain the observed NO homeostasis. Determinations of enzyme kinetic parameters in vivo are not trivial but evidently required to understand and model NO kinetics in denitrifying organisms in soils and aquatic environments.

SERR Spectroelectrochemical Study of Cytochrome cd1 Nitrite Reductase Co-Immobilized with Physiological Redox Partner Cytochrome c552 on Biocompatible Metal Electrodes.

Cytochrome cd1 nitrite reductases (cd 1NiRs) catalyze the one-electron reduction of nitrite to nitric oxide. Due to their catalytic reaction, cd 1NiRs are regarded as promising components for biosensing, bioremediation and biotechnological applications. Motivated by earlier findings that catalytic activity of cd 1NiR from Marinobacter hydrocarbonoclasticus (Mhcd 1) depends on the presence of its physiological redox partner, cytochrome c 552 (cyt c 552), we show here a detailed surface enhanced resonance Raman characterization of Mhcd 1 and cyt c 552 attached to biocompatible electrodes in conditions which allow direct electron transfer between the conducting support and immobilized proteins. Mhcd 1 and cyt c552 are co-immobilized on silver electrodes coated with self-assembled monolayers (SAMs) and the electrocatalytic activity of Ag // SAM // Mhcd 1 // cyt c 552 and Ag // SAM // cyt c 552 // Mhcd 1 constructs is tested in the presence of nitrite. Simultaneous evaluation of structural and thermodynamic properties of the immobilized proteins reveals that cyt c 552 retains its native properties, while the redox potential of apparently intact Mhcd 1 undergoes a ~150 mV negative shift upon adsorption. Neither of the immobilization strategies results in an active Mhcd 1, reinforcing the idea that subtle and very specific interactions between Mhcd 1 and cyt c 552 govern efficient intermolecular electron transfer and catalytic activity of Mhcd 1.

Response of denitrifying genes coding for nitrite (nirK or nirS) and nitrous oxide (nosZ) reductases to different physico-chemical parameters during agricultural waste composting

The present research was performed to clarify the changes of denitrifying genes (nirK, nirS, and nosZ) abundances under different physico-chemical parameters through evaluating the relationships between the genes abundances and parameters during agricultural waste composting. The genes abundances were determined by real-time quantitative PCR (qPCR). The correlations between physico-chemical parameters and denitrifying genes abundances were analysed by regression analysis. qPCR results showed that the nosZ gene abundance was higher than that of nirK and nirS genes. The nirK gene abundance was higher than nirS gene indicating that nitrite reducers with Cu-containing enzyme encoded by nirK gene were more of importance than those with cytochrome cd1 nitrite reductase encoded by nirS gene in the nitrite reduction step. Regression analysis suggested that (1) nirK gene abundance was correlated with pile temperature following quadratic model; (2) nirS gene abundance was linearly correlated with pile temperature and concentration of NH4 (+), while correlated with concentration of NO3 (-) and pH following inverse and quadratic model respectively; (3) nosZ gene abundance was quadratically correlated with pH and linearly correlated with water soluble carbon (WSC).

Electron donors and co-contaminants affect microbial community composition and activity in perchlorate degradation.

Although microbial reduction of perchlorate (ClO4(-)) is a promising and effective method, our knowledge on the changes in microbial communities during ClO4(-) degradation is limited, especially when different electron donors are supplied and/or other contaminants are present. Here, we examined the effects of acetate and hydrogen as electron donors and nitrate and ammonium as co-contaminants on ClO4(-) degradation by anaerobic microcosms using six treatments. The process of degradation was divided into the lag stage (SI) and the accelerated stage (SII). Quantitative PCR was used to quantify four genes: pcrA (encoding perchlorate reductase), cld (encoding chlorite dismutase), nirS (encoding copper and cytochrome cd1 nitrite reductase), and 16S rRNA. While the degradation of ClO4(-) with acetate, nitrate, and ammonia system (PNA) was the fastest with the highest abundance of the four genes, it was the slowest in the autotrophic system (HYP). The pcrA gene accumulated in SI and played a key role in initiating the accelerated degradation of ClO4(-) when its abundance reached a peak. Degradation in SII was primarily maintained by the cld gene. Acetate inhibited the growth of perchlorate-reducing bacteria (PRB), but its effect was weakened by nitrate (NO3(-)), which promoted the growth of PRB in SI, and therefore, accelerated the ClO4(-) degradation rate. In addition, ammonia (NH4(+)), as nitrogen sources, accelerated the growth of PRB. The bacterial communities' structure and diversity were significantly affected by electron donors and co-contaminants. Under heterotrophic conditions, both ammonia and nitrate promoted Azospira as the most dominant genera, a fact that might significantly influence the rate of ClO4(-) natural attenuation by degradation.

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.

The Crystal Structure of Siroheme Decarboxylase in Complex with Iron-Uroporphyrin III Reveals Two Essential Histidine Residues

The isobacteriochlorin heme d1 serves as an essential cofactor in the cytochrome cd1 nitrite reductase NirS that plays an important role for denitrification. During the biosynthesis of heme d1, the enzyme siroheme decarboxylase catalyzes the conversion of siroheme to 12,18-didecarboxysiroheme. This enzyme was discovered recently (Bali S, Lawrence AD, Lobo SA, Saraiva LM, Golding BT, Palmer DJ et al. Molecular hijacking of siroheme for the synthesis of heme and d1 heme. Proc Natl Acad Sci USA 2011;108:18260–5) and is only scarcely characterized. Here, we present the crystal structure of the siroheme decarboxylase from Hydrogenobacter thermophilus representing the first three-dimensional structure for this type of enzyme. The overall structure strikingly resembles those of transcriptional regulators of the Lrp/AsnC family. Moreover, the structure of the enzyme in complex with a substrate analog reveals first insights into its active-site architecture. Through site-directed mutagenesis and subsequent biochemical characterization of the enzyme variants, two conserved histidine residues within the active site are identified to be involved in substrate binding and catalysis. Based on our results, we propose a potential catalytic mechanism for the enzymatic reaction catalyzed by the siroheme decarboxylase.

Reducing NO and N₂O emission during aerobic denitrification by newly isolated Pseudomonas stutzeri PCN-1.

As two obligatory intermediates of denitrification, both NO and N2O had harmful environmental and biological impacts. An aerobic denitrifying bacterial strain PCN-1 was newly isolated and identified as Pseudomonas stutzeri, which was capable of high efficient nitrogen removal under aerobic condition with maximal NO and N2O accumulation as low as 0.003% and 0.33% of removed NO3(-)-N, respectively. Further experiment taking nitrite as denitrifying substrate indicated similar low NO and N2O emission of 0.006% and 0.29% of reduced NO2(-)-N, respectively. Reverse transcription-polymerase chain reaction (RT-PCR) analysis revealed that the coordinate expression of denitrification gene nirS (for cytochrome cd1 nitrite reductase), cnorB (for NO reductase) and nosZ (for N2O reductase) was the fundamental reason of low NO and N2O accumulation. Activated sludge system bioaugmented by strain PCN-1 demonstrated a significant reduction of NO and N2O emission from wastewater during aerobic denitrification, implied great potential of PCN-1 in practical applications.

Maturation of the cytochrome cd1 nitrite reductase NirS from Pseudomonas aeruginosa requires transient interactions between the three proteins NirS, NirN and NirF

The periplasmic cytochrome cd1 nitrite reductase NirS occurring in denitrifying bacteria such as the human pathogen Pseudomonas aeruginosa contains the essential tetrapyrrole cofactors haem c and haem d1. Whereas the haem c is incorporated into NirS by the cytochrome c maturation system I, nothing is known about the insertion of the haem d1 into NirS. Here, we show by co-immunoprecipitation that NirS interacts with the potential haem d1 insertion protein NirN in vivo. This NirS–NirN interaction is dependent on the presence of the putative haem d1 biosynthesis enzyme NirF. Further, we show by affinity co-purification that NirS also directly interacts with NirF. Additionally, NirF is shown to be a membrane anchored lipoprotein in P. aeruginosa. Finally, the analysis by UV–visible absorption spectroscopy of the periplasmic protein fractions prepared from the P. aeruginosa WT (wild-type) and a P. aeruginosa ΔnirN mutant shows that the cofactor content of NirS is altered in the absence of NirN. Based on our results, we propose a potential model for the maturation of NirS in which the three proteins NirS, NirN and NirF form a transient, membrane-associated complex in order to achieve the last step of haem d1 biosynthesis and insertion of the cofactor into NirS.

Nitric oxide synthesis by bacterial cytochrome cd1 nitrite reductases

Cytochrome cd 1 nitrite reductase ( cd 1 NIR) is a haem-containing enzyme responsible for the reduction of nitrite into nitric oxide (NO), a key step in the bacterial anaerobic respiratory process of denitrification. The active site of cytochrome cd 1 contains the unique d 1 -haem cofactor, from which NO must be released. In general, reduced haems bind NO tightly relative to oxidised haems. The mechanism of NO release employed by cd 1 NiRs has been recently outlined, highlighting the essential role of the peculiar d 1 -haem, whose presence in these enzymes was largely unexplained. Briefly, we have shown that cd 1 NiRs can release quite rapidly the NO bound to the reduced d 1 haem. This finding challenged the classical idea that NO is often an “irreversible” inhibitor of reduced hemoproteins and prompted new studies on the reactivity of ferrous d 1 haem with NO, indicating that the d 1 haem has evolved to have low affinity for NO, as compared to other ferrous haems.

Benthic Nitrogen Loss in the Arabian Sea Off Pakistan

A pronounced deficit of nitrogen (N) in the oxygen minimum zone (OMZ) of the Arabian Sea suggests the occurrence of heavy N-loss that is commonly attributed to pelagic processes. However, the OMZ water is in direct contact with sediments on three sides of the basin. Contribution from benthic N-loss to the total N-loss in the Arabian Sea remains largely unassessed. In October 2007, we sampled the water column and surface sediments along a transect cross-cutting the Arabian Sea OMZ at the Pakistan continental margin, covering a range of station depths from 360 to 1430 m. Benthic denitrification and anammox rates were determined by using 15N-stable isotope pairing experiments. Intact core incubations showed declining rates of total benthic N-loss with water depth from 0.55 to 0.18 mmol N m−2 day−1. While denitrification rates measured in slurry incubations decreased from 2.73 to 1.46 mmol N m−2 day−1 with water depth, anammox rates increased from 0.21 to 0.89 mmol N m−2 day−1. Hence, the contribution from anammox to total benthic N-loss increased from 7% at 360 m to 40% at 1430 m. This trend is further supported by the quantification of cd1-containing nitrite reductase (nirS), the biomarker functional gene encoding for cytochrome cd1-Nir of microorganisms involved in both N-loss processes. Anammox-like nirS genes within the sediments increased in proportion to total nirS gene copies with water depth. Moreover, phylogenetic analyses of NirS revealed different communities of both denitrifying and anammox bacteria between shallow and deep stations. Together, rate measurement and nirS analyses showed that anammox, determined for the first time in the Arabian Sea sediments, is an important benthic N-loss process at the continental margin off Pakistan, especially in the sediments at deeper water depths. Extrapolation from the measured benthic N-loss to all shelf sediments within the basin suggests that benthic N-loss may be responsible for about half of the overall N-loss in the Arabian Sea.