Recent mechanistic developments for cytochrome c nitrite reductase, the key enzyme in the dissimilatory nitrate reduction to ammonium pathway.

Cytochrome c nitrite reductase, NrfA, is a soluble, periplasmic pentaheme cytochrome responsible for the reduction of nitrite to ammonium in the Dissimilatory Nitrate Reduction to Ammonium (DNRA) pathway, a vital reaction in the global nitrogen cycle. NrfA catalyzes this six-electron and eight-proton reduction of nitrite at a single active site with the help of its quinol oxidase partners. In this review, we summarize the latest progress in elucidating the reaction mechanism of ammonia production, including new findings about the active site architecture of NrfA, as well as recent results that elucidate electron transfer and storage in the pentaheme scaffold of this enzyme.

Trichlorobacter ammonificans, a dedicated acetate-dependent ammonifier with a novel module for dissimilatory nitrate reduction to ammonia.

Dissimilatory nitrate reduction to ammonia (DNRA) is a common biochemical process in the nitrogen cycle in natural and man-made habitats, but its significance in wastewater treatment plants is not well understood. Several ammonifying Trichlorobacter strains (former Geobacter) were previously enriched from activated sludge in nitrate-limited chemostats with acetate as electron (e) donor, demonstrating their presence in these systems. Here, we isolated and characterized the new species Trichlorobacter ammonificans strain G1 using a combination of low redox potential and copper-depleted conditions. This allowed purification of this DNRA organism from competing denitrifiers. T. ammonificans is an extremely specialized ammonifier, actively growing only with acetate as e-donor and carbon source and nitrate as e-acceptor, but H2 can be used as an additional e-donor. The genome of G1 does not encode the classical ammonifying modules NrfAH/NrfABCD. Instead, we identified a locus encoding a periplasmic nitrate reductase immediately followed by an octaheme cytochrome c that is conserved in many Geobacteraceae species. We purified this octaheme cytochrome c protein (TaNiR), which is a highly active dissimilatory ammonifying nitrite reductase loosely associated with the cytoplasmic membrane. It presumably interacts with two ferredoxin subunits (NapGH) that donate electrons from the menaquinol pool to the periplasmic nitrate reductase (NapAB) and TaNiR. Thus, the Nap-TaNiR complex represents a novel type of highly functional DNRA module. Our results indicate that DNRA catalyzed by octaheme nitrite reductases is a metabolic feature of many Geobacteraceae, representing important community members in various anaerobic systems, such as rice paddy soil and wastewater treatment facilities.

A Kinetic Investigation of the Early Steps in Cytochrome c Nitrite Reductase (ccNiR)-Catalyzed Reduction of Nitrite.

The decaheme enzyme cytochrome c nitrite reductase (ccNiR) catalyzes reduction of nitrite to ammonium in a six-electron, eight-proton process. With a strong reductant as the electron source, ammonium is the sole product. However, intermediates accumulate when weaker reductants are employed, facilitating study of the ccNiR mechanism. Herein, the early stages of Shewanella oneidensis ccNiR-catalyzed nitrite reduction were investigated by using the weak reductants N,N,N',N'-tetramethyl-p-phenylenediamine (TMPD) and ferrocyanide. In stopped-flow experiments, reduction of nitrite-loaded ccNiR by TMPD generated a transient intermediate, identified as FeH1II(NO2-), where FeH1 represents the ccNiR active site. FeH1II(NO2-) accumulated rapidly and was then more slowly converted to the two-electron-reduced moiety {FeH1NO}7; ccNiR was not reduced beyond the {FeH1NO}7 state. The midpoint potentials for sequential reduction of FeH1III(NO2-) to FeH1II(NO2-) and then to {FeH1NO}7 were estimated to be 130 and 370 mV versus the standard hydrogen electrode, respectively. FeH1II(NO2-) does not accumulate at equilibrium because its reduction to {FeH1NO}7 is so much easier than the reduction of FeH1III(NO2-) to FeH1II(NO2-). With weak reductants, free NO• was released from nitrite-loaded ccNiR. The release of NO• from {FeH1NO}7 is exceedingly slow (k ∼ 0.001 s-1), but it is somewhat faster (k ∼ 0.050 s-1) while FeH1III(NO2-) is being reduced to {FeH1NO}7; then, the release of NO• from the undetectable transient {FeH1NO}6 can compete with reduction of {FeH1NO}6 to {FeH1NO}7. CcNiR appears to be optimized to capture nitrite and minimize the release of free NO•. Nitrite capture is achieved by reducing bound nitrite with even weak electron donors, while NO• release is minimized by stabilizing the substitutionally inert {FeH1NO}7 over the more labile {FeH1NO}6.

Elucidating Electron Storage and Distribution within the Pentaheme Scaffold of Cytochrome c Nitrite Reductase (NrfA).

Cytochrome c nitrite reductases (CcNIR or NrfA) play important roles in the global nitrogen cycle by conserving the usable nitrogen in the soil. Here, the electron storage and distribution properties within the pentaheme scaffold of Geobacter lovleyi NrfA were investigated via electron paramagnetic resonance (EPR) spectroscopy coupled with chemical titration experiments. Initially, a chemical reduction method was established to sequentially add electrons to the fully oxidized protein, 1 equiv at a time. The step-by-step reduction of the hemes was then followed using ultraviolet-visible absorption and EPR spectroscopy. EPR spectral simulations were used to elucidate the sequence of heme reduction within the pentaheme scaffold of NrfA and identify the signals of all five hemes in the EPR spectra. Electrochemical experiments ascertain the reduction potentials for each heme, observed in a narrow range from +10 mV (heme 5) to -226 mV (heme 3) (vs the standard hydrogen electrode). On the basis of quantitative analysis and simulation of the EPR data, we demonstrate that hemes 4 and 5 are reduced first (before the active site heme 1) and serve the purpose of an electron storage unit within the protein. To probe the role of the central heme 3, an H108M NrfA variant was generated where the reduction potential of heme 3 is shifted positively (from -226 to +48 mV). The H108M mutation significantly impacts the distribution of electrons within the pentaheme scaffold and the reduction potentials of the hemes, reducing the catalytic activity of the enzyme to 1% compared to that of the wild type. We propose that this is due to heme 3's important role as an electron gateway in the wild-type enzyme.

Cytochrome c nitrite reductase from the bacterium Geobacter lovleyi represents a new NrfA subclass.

Cytochrome c nitrite reductase (NrfA) catalyzes the reduction of nitrite to ammonium in the dissimilatory nitrate reduction to ammonium (DNRA) pathway, a process that competes with denitrification, conserves nitrogen, and minimizes nutrient loss in soils. The environmental bacterium Geobacter lovleyi has recently been recognized as a key driver of DNRA in nature, but its enzymatic pathway is still uncharacterized. To address this limitation, here we overexpressed, purified, and characterized G. lovleyi NrfA. We observed that the enzyme crystallizes as a dimer but remains monomeric in solution. Importantly, its crystal structure at 2.55-Å resolution revealed the presence of an arginine residue in the region otherwise occupied by calcium in canonical NrfA enzymes. The presence of EDTA did not affect the activity of G. lovleyi NrfA, and site-directed mutagenesis of this arginine reduced enzymatic activity to <3% of the WT levels. Phylogenetic analysis revealed four separate emergences of Arg-containing NrfA enzymes. Thus, the Ca2+-independent, Arg-containing NrfA from G. lovleyi represents a new subclass of cytochrome c nitrite reductase. Most genera from the exclusive clades of Arg-containing NrfA proteins are also represented in clades containing Ca2+-dependent enzymes, suggesting convergent evolution.

Spontaneous Mutations in the Nitrate Reductase Gene napC Drive the Emergence of Eco-friendly Low-N2O-Emitting Alfalfa Rhizobia in Regions with Different Climates.

We have recently shown that commercial alfalfa inoculants (e.g., Sinorhizobium meliloti B399), which are closely related to the denitrifier model strain Sinorhizobium meliloti 1021, have conserved nitrate, nitrite, and nitric oxide reductases associated with the production of the greenhouse gas nitrous oxide (N2O) from nitrate but lost the N2O reductase related to the degradation of N2O to gas nitrogen. Here, we screened a library of nitrogen-fixing alfalfa symbionts originating from different ecoregions and containing N2O reductase genes and identified novel rhizobia (Sinorhizobium meliloti INTA1-6) exhibiting exceptionally low N2O emissions. To understand the genetic basis of this novel eco-friendly phenotype, we sequenced and analyzed the genomes of these strains, focusing on their denitrification genes, and found mutations only in the nitrate reductase structural gene napC. The evolutionary analysis supported that, in these natural strains, the denitrification genes were inherited by vertical transfer and that their defective nitrate reductase napC alleles emerged by independent spontaneous mutations. In silico analyses showed that mutations in this gene occurred in ssDNA loop structures with high negative free energy (-ΔG) and that the resulting mutated stem-loop structures exhibited increased stability, suggesting the occurrence of transcription-associated mutation events. In vivo assays supported that at least one of these ssDNA sites is a mutational hot spot under denitrification conditions. Similar benefits from nitrogen fixation were observed when plants were inoculated with the commercial inoculant B399 and strains INTA4-6, suggesting that the low-N2O-emitting rhizobia can be an ecological alternative to the current inoculants without resigning economic profitability.

Trapping of a Putative Intermediate in the Cytochrome c Nitrite Reductase (ccNiR)-Catalyzed Reduction of Nitrite: Implications for the ccNiR Reaction Mechanism.

Cytochrome c nitrite reductase (ccNiR) is a periplasmic, decaheme homodimeric enzyme that catalyzes the six-electron reduction of nitrite to ammonia. Under standard assay conditions catalysis proceeds without detected intermediates, and it has been assumed that this is also true in vivo. However, this report demonstrates that it is possible to trap a putative intermediate by controlling the electrochemical potential at which reduction takes place. UV/vis spectropotentiometry showed that nitrite-loaded Shewanella oneidensis ccNiR is reduced in a concerted two-electron step to generate an {FeNO}7 moiety at the active site, with an associated midpoint potential of +246 mV vs SHE at pH 7. By contrast, cyanide-bound active site reduction is a one-electron process with a midpoint potential of +20 mV, and without a strong-field ligand the active site midpoint potential shifts 70 mV lower still. EPR analysis subsequently revealed that the {FeNO}7 moiety possesses an unusual spectral signature, different from those normally observed for {FeNO}7 hemes, that may indicate magnetic interaction of the active site with nearby hemes. Protein film voltammetry experiments previously showed that catalytic nitrite reduction to ammonia by S. oneidensis ccNiR requires an applied potential of at least -120 mV, well below the midpoint potential for {FeNO}7 formation. Thus, it appears that an {FeNO}7 active site is a catalytic intermediate in the ccNiR-mediated reduction of nitrite to ammonia, whose degree of accumulation depends exclusively on the applied potential. At low potentials the species is rapidly reduced and does not accumulate, while at higher potentials it is trapped, thus preventing catalytic ammonia formation.

Elucidating the Structures of the Low- and High-pH Mo(V) Species in Respiratory Nitrate Reductase: A Combined EPR, 14,15N HYSCORE, and DFT Study.

Respiratory nitrate reductases (Nars), members of the prokaryotic Mo/W-bis Pyranopterin Guanosine dinucleotide (Mo/W-bisPGD) enzyme superfamily, are key players in nitrate respiration, a major bioenergetic pathway widely used by microorganisms to cope with the absence of dioxygen. The two-electron reduction of nitrate to nitrite takes place at their active site, where the molybdenum ion cycles between Mo(VI) and Mo(IV) states via a Mo(V) intermediate. The active site shows two distinct pH-dependent Mo(V) electron paramagnetic resonance (EPR) signals whose structure and catalytic relevance have long been debated. In this study, we use EPR and HYSCORE techniques to probe their nuclear environment in Escherichia coli Nar (EcNar). By using samples prepared at different pH and through different enrichment strategies in 98Mo and 15N nuclei, we demonstrate that each of the two Mo(V) species is coupled to a single nitrogen nucleus with similar quadrupole characteristics. Structure-based density functional theory calculations allow us to propose a molecular model of the low-pH Mo(V) species consistent with EPR spectroscopic data. Our results show that the metal ion is coordinated by a monodentate aspartate ligand and permit the assignment of the coupled nitrogen nuclei to the Nδ of Asn52, a residue located ∼3.9 Å to the Mo atom in the crystal structures. This is confirmed by measurements on selectively 15N-Asn labeled EcNar. Further, we propose a Mo-O(H)···HN structure to account for the transfer of spin density onto the interacting nitrogen nucleus deduced from HYSCORE analysis. This work provides a foundation for monitoring the structure of the molybdenum active site in the presence of various substrates or inhibitors in Nars and other molybdenum enzymes.

Nitrate reduction in Haloferax alexandrinus: the case of assimilatory nitrate reductase.

Haloferax alexandrinus Strain TM JCM 10717T = IFO 16590T is an extreme halophilic archaeon able to produce significant amounts of canthaxanthin. Its genome sequence has been analysed in this work using bioinformatics tools available at Expasy in order to look for genes encoding nitrate reductase-like proteins: respiratory nitrate reductase (Nar) and/or assimilatory nitrate reductase (Nas). The ability of the cells to reduce nitrate under aerobic conditions was tested. The enzyme in charge of nitrate reduction under aerobic conditions (Nas) has been purified and characterised. It is a monomeric enzyme (72 ± 1.8 kDa) that requires high salt concentration for stability and activity. The optimum pH value for activity was 9.5. Effectiveness of different substrates, electron donors, cofactors and inhibitors was also reported. High nitrite concentrations were detected within the culture media during aerobic/microaerobic cells growth. The main conclusion from the results is that this haloarchaeon reduces nitrate aerobically thanks to Nas and may induce denitrification under anaerobic/microaerobic conditions using nitrate as electron acceptor. The study sheds light on the role played by haloarchaea in the biogeochemical cycle of nitrogen, paying special attention to nitrate reduction processes. Besides, it provides useful information for future attempts on microecological and biotechnological implications of haloarchaeal nitrate reductases.

A bioinspired iron catalyst for nitrate and perchlorate reduction.

Nitrate and perchlorate have considerable use in technology, synthetic materials, and agriculture; as a result, they have become pervasive water pollutants. Industrial strategies to chemically reduce these oxyanions often require the use of harsh conditions, but microorganisms can efficiently reduce them enzymatically. We developed an iron catalyst inspired by the active sites of nitrate reductase and (per)chlorate reductase enzymes. The catalyst features a secondary coordination sphere that aids in oxyanion deoxygenation. Upon reduction of the oxyanions, an iron(III)-oxo is formed, which in the presence of protons and electrons regenerates the catalyst and releases water.

In meso crystal structure of a novel membrane-associated octaheme cytochrome c from the Crenarchaeon Ignicoccus hospitalis.

The Crenarchaeon Ignicoccus hospitalis lives in symbiosis with Nanoarchaeum equitans providing essential cell components and nutrients to its symbiont. Ignicoccus hospitalis shows an intriguing morphology that points toward an evolutionary role in driving compartmentalization. Therefore, the bioenergetics of this archaeal host-symbiont system remains a pressing question. To date, the only electron acceptor described for I. hospitalis is elemental sulfur, but the organism comprises genes that encode for enzymes involved in nitrogen metabolism, e.g., one nitrate reductase and two octaheme cytochrome c, Igni_0955 (IhOCC) and Igni_1359. Herein, we detail functional and structural studies of the highly abundant IhOCC, including an X-ray crystal structure at 1.7 Å resolution, the first three-dimensional structure of an archaeal OCC. The trimeric IhOCC is membrane associated and exhibits significant structural and functional differences to previously characterized homologs within the hydroxylamine oxidoreductases (HAOs) and octaheme cytochrome c nitrite reductases (ONRs). The positions and spatial arrangement of the eight hemes are highly conserved, but the axial ligands of the individual hemes 3, 6 and 7 and the protein environment of the active site show significant differences. Most notably, the active site heme 4 lacks porphyrin-tyrosine cross-links present in the HAO family. We show that IhOCC efficiently reduces nitrite and hydroxylamine, with possible relevance to detoxification or energy conservation. DATABASE: Structural data are available in the Protein Data Bank under the accession number 4QO5.

Optimisation of nitrate reductase enzyme activity to synthesise silver nanoparticles.

Today, the synthesis of silver nanoparticles (Ag NPs) is very common since it has many applications in different areas. The synthesis of these nanoparticles is done by means of physical, chemical, or biological methods. However, due to its inexpensive and environmentally friendly features, the biological method is more preferable. In the present study, using nitrate reductase enzyme available in the Escherichia coli (E. coli) bacterium, the biosynthesis of Ag NPs was investigated. In addition, the activity of the nitrate reductase enzyme was optimised by changing its cultural conditions, and the effects of silver nitrate (AgNO(3)) concentration and enzyme amount on nanoparticles synthesis were studied. Finally, the produced nanoparticles were studied using ultraviolet -visible (UV-Vis) spectrophotometer, dynamic light scattering technique, and transmission electron microscopy. UV-Visible spectrophotometric study showed the characteristic peak for Ag NPs at wavelength 405-420 nm for 1 mM metal precursor solution (AgNO(3)) with 1, 5, 10, and 20 cc supernatant and 435 nm for 0.01M AgNO(3) with 20 cc supernatant. In this study, it was found that there is a direct relationship between the AgNO(3) concentration and the size of produced Ag NPs.

Identification of Amino Acids at the Catalytic Site of a Ferredoxin-Dependent Cyanobacterial Nitrate Reductase.

An in silico model of the ferredoxin-dependent nitrate reductase from the cyanobacterium Synechococcus sp. PCC 7942, and information about active sites in related enzymes, had identified Cys148, Met149, Met306, Asp163, and Arg351 as amino acids likely to be involved in either nitrate binding, prosthetic group binding, or catalysis. Site-directed mutagenesis was used to alter each of these residues, and differences in enzyme activity and substrate binding of the purified variants were analyzed. In addition, the effects of these replacements on the assembly and properties of the Mo cofactor and [4Fe-4S] centers were investigated using Mo and Fe determinations, coupled with electron paramagnetic resonance spectroscopy. The C148A, M149A, M306A, D163N, and R351Q variants were all inactive with either the physiological electron donor, reduced ferredoxin, or the nonphysiological electron donor, reduced methyl viologen, as the source of electrons, and all exhibited changes in the properties of the Mo cofactor. Charge-conserving D163E and R351K variants were also inactive, suggesting that specific amino acids are required at these two positions. The implications for the role of these five conserved active-site residues in light of these new results and previous structural, spectroscopic, and mutagenesis studies for related periplasmic nitrate reductases are discussed.

An ethane-bridged porphyrin dimer as a model of di-heme proteins: inorganic and bioinorganic perspectives and consequences of heme-heme interactions.

Interaction between heme centers has been cleverly implemented by Nature in order to regulate different properties of multiheme cytochromes, thereby allowing them to perform a wide variety of functions. Our broad interest lies in unmasking the roles played by heme-heme interactions in modulating different properties viz., metal spin state, redox potential etc., of the individual heme centers using an ethane-bridged porphyrin dimer as a synthetic model of dihemes. The large differences in the structure and properties of the diheme complexes, as compared to the monoheme analogs, provide unequivocal evidence of the role played by heme-heme interactions in the dihemes. This Perspective provides a brief account of our recent efforts to explore these interesting aspects and the subsequent outcomes.

Correlations between the Electronic Properties of Shewanella oneidensis Cytochrome c Nitrite Reductase (ccNiR) and Its Structure: Effects of Heme Oxidation State and Active Site Ligation.

The electrochemical properties of Shewanella oneidensis cytochrome c nitrite reductase (ccNiR), a homodimer that contains five hemes per protomer, were investigated by UV-visible and electron paramagnetic resonance (EPR) spectropotentiometries. Global analysis of the UV-vis spectropotentiometric results yielded highly reproducible values for the heme midpoint potentials. These midpoint potential values were then assigned to specific hemes in each protomer (as defined in previous X-ray diffraction studies) by comparing the EPR and UV-vis spectropotentiometric results, taking advantage of the high sensitivity of EPR spectra to the structural microenvironment of paramagnetic centers. Addition of the strong-field ligand cyanide led to a 70 mV positive shift of the active site's midpoint potential, as the cyanide bound to the initially five-coordinate high-spin heme and triggered a high-spin to low-spin transition. With cyanide present, three of the remaining hemes gave rise to distinctive and readily assignable EPR spectral changes upon reduction, while a fourth was EPR-silent. At high applied potentials, interpretation of the EPR spectra in the absence of cyanide was complicated by a magnetic interaction that appears to involve three of five hemes in each protomer. At lower applied potentials, the spectra recorded in the presence and absence of cyanide were similar, which aided global assignment of the signals. The midpoint potential of the EPR-silent heme could be assigned by default, but the assignment was also confirmed by UV-vis spectropotentiometric analysis of the H268M mutant of ccNiR, in which one of the EPR-silent heme's histidine axial ligands was replaced with a methionine.

Structural study of the X-ray-induced enzymatic reaction of octahaem cytochrome C nitrite reductase.

Octahaem cytochrome c nitrite reductase from the bacterium Thioalkalivibrio nitratireducens catalyzes the reduction of nitrite to ammonium and of sulfite to sulfide. The reducing properties of X-ray radiation and the high quality of the enzyme crystals allow study of the catalytic reaction of cytochrome c nitrite reductase directly in a crystal of the enzyme, with the reaction being induced by X-rays. Series of diffraction data sets with increasing absorbed dose were collected from crystals of the free form of the enzyme and its complexes with nitrite and sulfite. The corresponding structures revealed gradual changes associated with the reduction of the catalytic haems by X-rays. In the case of the nitrite complex the conversion of the nitrite ions bound in the active sites to NO species was observed, which is the beginning of the catalytic reaction. For the free form, an increase in the distance between the oxygen ligand bound to the catalytic haem and the iron ion of the haem took place. In the case of the sulfite complex no enzymatic reaction was detected, but there were changes in the arrangement of the active-site water molecules that were presumably associated with a change in the protonation state of the sulfite ions.

Construction of effective disposable biosensors for point of care testing of nitrite.

In this paper we aim to demonstrate, as a proof-of-concept, the feasibility of the mass production of effective point of care tests for nitrite quantification in environmental, food and clinical samples. Following our previous work on the development of third generation electrochemical biosensors based on the ammonia forming nitrite reductase (ccNiR), herein we reduced the size of the electrodes' system to a miniaturized format, solved the problem of oxygen interference and performed simple quantification assays in real samples. In particular, carbon paste screen printed electrodes (SPE) were coated with a ccNiR/carbon ink composite homogenized in organic solvents and cured at low temperatures. The biocompatibility of these chemical and thermal treatments was evaluated by cyclic voltammetry showing that the catalytic performance was higher with the combination acetone and a 40°C curing temperature. The successful incorporation of the protein in the carbon ink/solvent composite, while remaining catalytically competent, attests for ccNiR's robustness and suitability for application in screen printed based biosensors. Because the direct electrochemical reduction of molecular oxygen occurs when electroanalytical measurements are performed at the negative potentials required to activate ccNiR (ca.-0.4V vs Ag/AgCl), an oxygen scavenging system based on the coupling of glucose oxidase and catalase activities was successfully used. This enabled the quantification of nitrite in different samples (milk, water, plasma and urine) in a straightforward way and with small error (1-6%). The sensitivity of the biosensor towards nitrite reduction under optimized conditions was 0.55 A M(-1) cm(-2) with a linear response range 0.7-370 µM.

Resolution of key roles for the distal pocket histidine in cytochrome C nitrite reductases.

Cytochrome c nitrite reductases perform a key step in the biogeochemical N-cycle by catalyzing the six-electron reduction of nitrite to ammonium. These multiheme cytochromes contain a number of His/His ligated c-hemes for electron transfer and a structurally differentiated heme that provides the catalytic center. The catalytic heme has proximal ligation from lysine, or histidine, and an exchangeable distal ligand bound within a pocket that includes a conserved histidine. Here we describe properties of a penta-heme cytochrome c nitrite reductase in which the distal His has been substituted by Asn. The variant is unable to catalyze nitrite reduction despite retaining the ability to reduce a proposed intermediate in that process, namely, hydroxylamine. A combination of electrochemical, structural and spectroscopic studies reveals that the variant enzyme simultaneously binds nitrite and electrons at the catalytic heme. As a consequence the distal His is proposed to play a key role in orienting the nitrite for N-O bond cleavage. The electrochemical experiments also reveal that the distal His facilitates rapid nitrite binding to the catalytic heme of the native enzyme. Finally it is noted that the thermodynamic descriptions of nitrite- and electron-binding to the active site of the variant enzyme are modulated by the prevailing oxidation states of the His/His ligated hemes. This behavior is likely to be displayed by other multicentered redox enzymes such that there are wide implications for considering the determinants of catalytic activity in this important and varied group of oxidoreductases.

Nitrite reduction by molybdoenzymes: a new class of nitric oxide-forming nitrite reductases.

Nitric oxide (NO) is a signalling molecule involved in several physiological processes, in both prokaryotes and eukaryotes, and nitrite is being recognised as an NO source particularly relevant to cell signalling and survival under challenging conditions. The "non-respiratory" nitrite reduction to NO is carried out by "non-dedicated" nitrite reductases, making use of metalloproteins present in cells to carry out other functions, such as several molybdoenzymes (a new class of nitric oxide-forming nitrite reductases). This minireview will highlight the physiological relevance of molybdenum-dependent nitrite-derived NO formation in mammalian, plant and bacterial signalling (and other) pathways. The mammalian xanthine oxidase/xanthine dehydrogenase, aldehyde oxidase, mitochondrial amidoxime-reducing component, plant nitrate reductase and bacterial aldehyde oxidoreductase and nitrate reductases will be considered. The nitrite reductase activity of each molybdoenzyme will be described and the review will be oriented to discuss the feasibility of the reactions from a (bio)chemical point of view. In addition, the molecular mechanism proposed for the molybdenum-dependent nitrite reduction will be discussed in detail.

Hydrogen bonding networks tune proton-coupled redox steps during the enzymatic six-electron conversion of nitrite to ammonia.

Multielectron multiproton reactions play an important role in both biological systems and chemical reactions involved in energy storage and manipulation. A key strategy employed by nature in achieving such complex chemistry is the use of proton-coupled redox steps. Cytochrome c nitrite reductase (ccNiR) catalyzes the six-electron seven-proton reduction of nitrite to ammonia. While a catalytic mechanism for ccNiR has been proposed on the basis of studies combining computation and crystallography, there have been few studies directly addressing the nature of the proton-coupled events that are predicted to occur along the nitrite reduction pathway. Here we use protein film voltammetry to directly interrogate the proton-coupled steps that occur during nitrite reduction by ccNiR. We find that conversion of nitrite to ammonia by ccNiR adsorbed to graphite electrodes is defined by two distinct phases; one is proton-coupled, and the other is not. Mutation of key active site residues (H257, R103, and Y206) modulates these phases and specifically alters the properties of the detected proton-dependent step but does not inhibit the ability of ccNiR to conduct the full reduction of nitrite to ammonia. We conclude that the active site residues examined are responsible for tuning the protonation steps that occur during catalysis, likely through an extensive hydrogen bonding network, but are not necessarily required for the reaction to proceed. These results provide important insight into how enzymes can specifically tune proton- and electron transfer steps to achieve high turnover numbers in a physiological pH range.