Health, Safety & Environment

Nitrate reductase

Biological nitrogen fixation can be catalysed by three isozymes of nitrogenase: molybdenum (Mo)-nitrogenase, vanadium (V)-nitrogenase and iron-only (Fe)-nitrogenase. The activity of these isozymes strongly depends on their metal cofactors, molybdenum, vanadium and iron, and their bioavailability in ecosystems.

Here, we show how metal bioavailability can be affected by the presence of tannic acid (organic matter), and the subsequent consequences on diazotrophic growth of the soil bacterium Azotobacter vinelandii.

In the presence of tannic acids, A. vinelandii produces a higher amount of metallophores, which coincides with an active, regulated and concomitant acquisition of molybdenum and vanadium under cellular conditions that are usually considered not molybdenum limiting. The associated nitrogenase genes exhibit decreased nifD expression and increased vnfD expression. Thus, in limiting bioavailable metal conditions, A. vinelandii takes advantage of its nitrogenase diversity to ensure optimal diazotrophic growth.

Jouogo Noumsi, C., Pourhassan, N., Darnajoux, R., Deicke, M., Wichard, T., Burrus, V., and Bellenger, J. P.,Effect of organic matter on nitrogenase metal cofactors homeostasis in Azotobacter vinelandii under diazotrophic conditions, Environmental microbiology reports, 2016, 8, 76-84.

NITRATE REDUCTASE

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 Ndelta of Asn52, a residue located approximately 3.9 A 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.

Rendon, J., Biaso, F., Ceccaldi, P., Toci, R., Seduk, F., Magalon, A., Guigliarelli, B., and Grimaldi, S.,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, Inorganic chemistry, 2017.

A dual system formed by the ARC and NR molybdoenzymes mediates nitrite-dependent NO production in Chlamydomonas

Nitric oxide (NO) is a relevant signal molecule involved in many plant processes. However, the mechanisms and proteins responsible for its synthesis are scarcely known. In most photosynthetic organisms NO synthases have not been identified, and Nitrate Reductase (NR) has been proposed as the main enzymatic NO source, a process that in vitro is also catalysed by other molybdoenzymes. By studying transcriptional regulation, enzyme approaches, activity assays with in vitro purified proteins and in vivo and in vitro NO determinations, we have addressed the role of NR and Amidoxime Reducing Component (ARC) in the NO synthesis process. NR and ARC were intimately related both at transcriptional and activity level. Thus, arc mutants showed high NIA1 (NR gene) expression and NR activity. Conversely, mutants without active NR displayed an increased ARC expression in nitrite medium. Our results with nia1 and arc mutants and with purified enzymes support that ARC catalyses the NO production from nitrite taking electrons from NR and not from Cytb5-1/Cytb5-Reductase, the component partners previously described for ARC (proposed as NOFNiR, Nitric Oxide-Forming Nitrite Reductase). This NR-ARC dual system would be able to produce NO in the presence of nitrate, condition under which NR is unable to do it.

Chamizo-Ampudia, A., Sanz-Luque, E., Llamas, A., Ocana-Calahorro, F., Mariscal, V., Carreras, A., Barroso, J. B., Galvan, A., and Fernandez, E.,A dual system formed by the ARC and NR molybdoenzymes mediates nitrite-dependent NO production in Chlamydomonas, Plant, cell & environment, 2016, 39, 2097-107.

Structural and mechanistic insights on nitrate reductases

Nitrate reductases (NR) belong to the DMSO reductase family of Mo-containing enzymes and perform key roles in the metabolism of the nitrogen cycle, reducing nitrate to nitrite. Due to variable cell location, structure and function, they have been divided into periplasmic (Nap), cytoplasmic, and membrane-bound (Nar) nitrate reductases. The first crystal structure obtained for a NR was that of the monomeric NapA from Desulfovibrio desulfuricans in 1999. Since then several new crystal structures were solved providing novel insights that led to the revision of the commonly accepted reaction mechanism for periplasmic nitrate reductases. The two crystal structures available for the NarGHI protein are from the same organism (Escherichia coli) and the combination with electrochemical and spectroscopic studies also lead to the proposal of a reaction mechanism for this group of enzymes. Here we present an overview on the current advances in structural and functional aspects of bacterial nitrate reductases, focusing on the mechanistic implications drawn from the crystallographic data.

Coelho, C., and Romao, M. J.,Structural and mechanistic insights on nitrate reductases, Protein Science, 2015, 24, 1901-1911.

Nitrate reductases activity, biomass, yield, and quality in cotton in response to nitrogen fertilization

In the production of cotton (Gossypium hirsutum L.), nitrogen fertilization is one of the most costly crop practices, but important to reach high yields. However, high nitrogen (N) content in plants does not always translate into a high fibre production. One way of assessing the efficiency of the N fertilizer is through the enzymatic activity of the nitrate reductase (NR). This is a key enzyme in N assimilation, whose activity is regulated by a number of endogenous and exogenous factors that determine yield. The aim of this study was to assess the effect of N fertilization on yield, fibre quality, biomass, and NR enzymatic activity in vivo in the cotton variety Fiber Max 989. The evaluated application rates were 0, 50, 100, and 150 kg/ha of N, using urea as a source (46% N) in a randomized-block design with three replicates. At harvest, the maximum yield of seed cotton and the greatest accumulation of total foliar biomass through time was reached after applying 150 kg N/ha. The different N-application rates did not affect the components of cotton-fibre quality. The activity of endogenous NR was greater on plants where 150 kg N/ha were applied. The highest cotton yield and N contents were obtained on these plants. Therefore, the NR activity in vivo could be used as a bioindicator of the N nutritional level in cotton.

Hernandez-Cruz, A. E., Sanchez, E., Preciado-Rangel, P., Garcia-Banuelos, M. L., Palomo-Gil, A., and Espinoza-Banda, A.,Nitrate reductase activity, biomass, yield, and quality in cotton in response to nitrogen fertilization, Phyton-International Journal of Experimental Botany, 2015, 84, 454-460.

Effect of molybdenum treatment on molybdenum concentration and nitrate reduction in maize seedlings

Since 1940 molybdenum has been known as an essential trace element in plant nutrition and physiology. It has a central role in nitrogen metabolism, and its deficiency leads to nitrate accumulation in plants. In this study, we cultivated maize seedlings (Zea mays L. cv. Norma SC) in nutrient solution and soil (rhizoboxes) to investigate the effect of molybdenum treatment on the absorption of molybdenum, sulfur and iron. These elements have been previously shown to play important roles in nitrate reduction, because they are necessary for the function of the nitrate reductase enzyme. We also investigated the relationship between molybdenum treatments and different nitrogen forms in maize. Molybdenum treatments were 0, 0.96, 9.6 and 96 mug kg-1in the nutrition solution experiments, and 0, 30, 90, 270 mg kg-1 in the rhizobox experiments. On the basis of our results, the increased Mo level produced higher plant available Mo concentration in nutrient solution and in soil, which resulted increased concentration of Mo in shoots and roots of maize seedlings. In addition it was observed that maize seedlings accumulated more molybdenum in their roots than in their shoots at all treatments. In contrast, molybdenum treatments did not affect significantly either iron or sulfur concentrations in the plant, even if these elements (Mo, S and Fe) play alike important roles in nitrogen metabolism. Furthermore, the physiological molybdenum level (1x Mo = 0.01 muM) reduced NO3-N and enhanced the NH4-N concentrations in seedlings, suggesting that nitrate reduction was more intense under a well-balanced molybdenum supply.

Kovacs, B., Puskas-Preszner, A., Huzsvai, L., Levai, L., and Bodi, E.,Effect of molybdenum treatment on molybdenum concentration and nitrate reduction in maize seedlings, Plant physiology and biochemistry : PPB / Societe francaise de physiologie vegetale, 2015, 96, 38-44.

Pyranopterin coordination controls molybdenum electrochemistry in escherichia coli nitrate reductase

We test the hypothesis that pyranopterin (PPT) coordination plays a critical role in defining molybdenum active site redox chemistry and reactivity in the mononuclear molybdoenzymes. The molybdenum atom of Escherichia coli nitrate reductase A (NarGHI) is coordinated by two PPT-dithiolene chelates that are defined as proximal and distal based on their proximity to a [4Fe-4S] cluster known as FS0. We examined variants of two sets of residues involved in PPT coordination: (I) those interacting directly or indirectly with the pyran oxygen of the bicyclic distal PPT (NarG-Ser(719), NarG-His(1163), and NarG-His(1184)); and (ii) those involved in bridging the two PPTs and stabilizing the oxidation state of the proximal PPT (NarG-His(1092) and NarGHis(1098)). A S719A variant has essentially no effect on the overall Mo(VI/IV) reduction potential, whereas the H1163A and H1184A variants elicit large effects (Delta E-m values of -88 and -36 mV, respectively). Ala variants of His(1092) and His(1098) also elicit large Delta E-m values of -143 and -101 mV, respectively. An Arg variant of His(1092) elicits a small Delta E-m of +18 mV on the Mo(VI/IV) reduction potential. There is a linear correlation between the molybdenum E-m value and both enzyme activity and the ability to support anaerobic respiratory growth on nitrate. These data support a non-innocent role for the PPT moieties in controlling active site metal redox chemistry and catalysis.

Wu, S. Y., Rothery, R. A., and Weiner, J. H.,Pyranopterin Coordination Controls Molybdenum Electrochemistry in Escherichia coli Nitrate Reductase, Journal of Biological Chemistry, 2015, 290, 25164-25173.

Structural and mechanistic insights on nitrate reductases

Nitrate reductases (NR) belong to the DMSO reductase family of Mo-containing enzymes and perform key roles in the metabolism of the nitrogen cycle, reducing nitrate to nitrite. Due to variable cell location, structure and function, they have been divided into periplasmic (Nap), cytoplasmic, and membrane-bound (Nar) nitrate reductases. The first crystal structure obtained for a NR was that of the monomeric NapA from Desulfovibrio desulfuricans in 1999. Since then several new crystal structures were solved providing novel insights that led to the revision of the commonly accepted reaction mechanism for periplasmic nitrate reductases. The two crystal structures available for the NarGHI protein are from the same organism (Escherichia coli) and the combination with electrochemical and spectroscopic studies also lead to the proposal of a reaction mechanism for this group of enzymes. Here we present an overview on the current advances in structural and functional aspects of bacterial nitrate reductases, focusing on the mechanistic implications drawn from the crystallographic data.

Coelho, C., and Romao, M. J.,Structural and mechanistic insights on nitrate reductases, Protein science : a publication of the Protein Society, 2015.

In plants and some animals the first stage in the reduction of nitrate is to nitrite. The reduction is catalysed by nitrate reductase, a flavoprotein enzyme which has molybdenum as the only metal requirement. Molybdenum acts as an electron acceptor from reduced FAD in the enzyme. The molybdenum cofactor is an oxomolybdenum sulfur species with a pterin ligand.

Berks, B. C., Ferguson, S. J., Moir, J. W. B. and Richardson, D. J., Biochim. Biophys. Acta - Bioenergetics , 1995, 1232, 97.
Campbell, W. H., Plant Physiology , 1996, 111 , 355.
Collison, D., Garner, C. D. and Joule, J. A., Chem. Soc. Rev. , 1996, 25 , 25.
Although NR always catalyzes the conversion of nitrate to nitrite, its location in the cell, structure, and function are organism-dependent. Protein sequencing is used to determine phylogenetic relationships and to examine similarities in structure and function. Conserved binding sites for molybdenum and pterin cofactors are found.

Stolz, J.F. and Basu, P., Evolution of nitrate reductase: Molecular and structural variations on a common function, Chembiochem, 2002, 3, 198-206.

Gonzalez, P. J., Correia, C., Moura, I., Brondino, C. D., and Moura, J. J. G., Bacterial nitrate reductases: Molecular and biological aspects of nitrate reduction, Journal of Inorganic Biochemistry, 2006, 100, 1015-1023.

Enzymes of the DMSO reductase family use a mononuclear Mo-bis(molybdopterin) cofactor (MoCo) to catalyze a variety of oxo-transfer reactions. Site-directed mutagenesis, EPR and protein film voltammetry were used to demonstrate that the MoCo in R. sphaeroides periplasmic nitrate reductase (NapAB) is subject to an irreversible reductive activation process. This activation quantitatively correlates with the disappearance of the so-called "Mo(V) high-g" EPR signal, but this reductive process is too slow to be part of the normal catalytic cycle. Therefore, in NapAB, this most intense and most commonly observed signature of the MoCo arises from a dead-end, inactive state that gives a catalytically competent species only after reduction. This activation proceeds, even without substrate, according to a reduction followed by an irreversible nonredox step, both of which are pH independent. An apparently similar process occurs in other nitrate reductases (both assimilatory and membrane bound), and this also recalls the redox cycling procedure, which activates periplasmic DMSO reductases and simplifies their spectroscopic signatures.

It is proposed that heterogeneity at the active site and reductive activation are common properties of enzymes from the DMSO reductase family.

Regarding NapAB, that no Mo EPR signal could be detected upon reoxidizing the fully reduced enzyme suggests that the catalytically active form of the Mo(V) is thermodynamically unstable, as for other enzymes of the DMSO reductase family.

Fourmond, V., Burlat, B., Dementin, S., Arnoux, P., Sabaty, M., Boiry, S., Guigliarelli, B., Bertrand, P., Pignol, D., and Leger, C., Major Mo(V) EPR Signature of Rhodobacter sphaeroides Periplasmic Nitrate Reductase Arising from a Dead-End Species That Activates upon Reduction. Relation to Other Molybdoenzymes from the DMSO Reductase Family, Journal of Physical Chemistry B, 2008, 112, 15478-15486.

Nitrate reductase in winter wheat enhanced by molybdenum

The objective was to study whether the accumulation and utilization of plant nitrogen are controlled by molybdenum status in winter wheat cultivars. Mo-efficient cultivar 97003 and Mo-inefficient cultivar 97014 were grown in severely Mo-deficient acidic soil (0.112 mg Mo kg-1) with and without the application of 0.13 mg Mo kg-1. The accumulation and use efficiency of plant total nitrogen were higher in the molybdenum-treated soil. The overall activity of nitrate reductase was higher in the molybdenum-treated soil and the activity of glutamine synthetase was lower. Concentration of nitrate and glutamate were also lower in the molybdenum-treated soil; evidences for enhanced nitgrogen use efficiency due to added molybdenum. Molybdenumd promote nitrogen accumulation and utilization in winter wheat which was directly related to nitrate reductase and feedback regulated by glutamine synthetase. Higher molybdenum status also results in higher accumulation and utilization of plant nitrogen in the Mo-efficient cultivar.

Yu, M., Hu, C. X., Sun, X. C., and Wang, Y. H., Influences of Mo on Nitrate Reductase, Glutamine Synthetase and Nitrogen Accumulation and Utilization in Mo-Efficient and Mo-Inefficient Winter Wheat Cultivars, Agricultural Sciences in China, 2010, 9, 355-361.

Nitrate reductase mechanism

Calculations show that the reduction of nitrate to nitrite can occur through an association of nitrate to the Mo center, followed by rupture of the Mo-O-NO2- bond. the direct coordination of nitrate to Mo is an almost barrier-free process, and the barrier for the rate-determining step of the Mo-O-NO2- bond cleavage is about 11.7 kcal mol-1, significantly lower than those in other plausible mechanisms. The presence of a disulfide bond in the active site can influence the interconversion of Mo(IV) to Mo(VI).

Xie, H. J. and Cao, Z. X., Enzymatic Reduction of Nitrate to Nitrite: Insight from Density Functional Calculations, Organometallics, 2010, 29, 436-441.

Nitrate reductase: Nitrate reductase from triticum aestivum lLeaves: Regulation of activity and possible role in production of nitric oxide

Nitrate reductase (NR) and peroxidase (POX) are important enzymes involved in the metabolism of reactive oxygen (ROS) and nitrogen species in leaves of wheat (Triticum aestivum L.) seedlings. It has been confirmed that NR activity in wheat leaves depends on the light conditions and the presence of nitrates during the cultivation of the seedlings, and it is regulated by the molybdenum cofactor and phosphorylation.

In the present study, confocal microscopy and EPR spectroscopy studies showed that the addition of nitrite, a product of NR, increased the level of nitric oxide (NO). This increase was prevented by the addition of sodium azide, an inhibitor of NR.

The results suggest that in wheat leaves one of the key functions of NR is the formation of the signaling NO molecule.

Cultivation of green plants under conditions of prolonged (4 days) darkness, a strong stress factor for photosynthesizing cells, decreased the activity of NR. Moreover, darkness induced significant elevation of the POX activity that was prevented by the addition of nitrate to the growth medium. It is proposed that the changes in light conditions result in the competition between nitrate- and ROS-metabolizing activities of POX in leaves, and a possible interaction between NR and POX controls the levels of NO and ROS in the leaf tissue

Galeeva, E. I., Trifonova, T. V., Ponomareva, A. A., Viktorova, L. V., and Minibayeva, F. V., Nitrate Reductase from Triticum aestivum Leaves: Regulation of Activity and Possible Role in Production of Nitric Oxide, Biochemistry-Moscow, 2012, 77, 404-410.

Nitrate reductase: DFT investigation of the molybdenum cofactor in periplasmic nitrate reductases: structure of the Mo(V) EPR-active species

The periplasmic nitrate reductase NAP belongs to the DMSO reductase family that regroups molybdoenzymes housing a bis-molybdopterin cofactor as the active site. Several forms of the Mo(V) state, an intermediate redox state in the catalytic cycle of the enzyme, have been evidenced by EPR spectroscopy under various conditions, but their structure and catalytic relevance are not fully understood.

On the basis of structural data available from the literature, we built several models that reproduce the first coordination sphere of the molybdenum cofactor and used DFT methods to make magneto-structural correlations on EPR-detected species.

"High-g" states, which are the most abundant Mo(V) species, are characterized by a low-anisotropy g tensor and a high g(min), value. We assign this signature to a six-sulfur coordination sphere in a pseudotrigonal prismatic geometry with a partial disulfide bond.

The "very high-g" species is well described with a sulfido ion as the sixth ligand.

The "low-g" signal can be successfully associated to a Mo(V) sulfite oxidase-type active site with only one pterin moiety coordinated to the molybdenum ion with an oxo or sulfido axial ligand.

For all these species we investigate their catalytic activity using a thermodynamic point of view on the molybdenum coordination sphere.

Beyond the periplasmic nitrate reductase case, this work provides useful magneto-structural correlations to characterize EPR-detected species in mononuclear molybdoenzymes

Biaso, Frederic, Burlat, Benedicte, and Guigliarelli, Bruno, DFT Investigation of the Molybdenum Cofactor in Periplasmic Nitrate Reductases: Structure of the Mo(V) EPR-Active Species, Inorganic Chemistry, 2012, 51, 3409-3419.

Nitrite reduction and cardiovascular protection

Inorganic nitrite, a metabolite of endogenously produced nitric oxide (NO) from NO synthases (NOS), provides the largest endocrine source of directly bioavailable NO. The conversion of nitrite to NO occurs mainly through enzymatic reduction, mediated by a range of proteins, including haem-globins, molybdo-flavoproteins, mitochondrial proteins, cytochrome P450 enzymes, and NOS. Such nitrite reduction is particularly favoured under hypoxia, when endogenous formation of NO from NOS is impaired. Under normoxic conditions, the majority of these nitrite reductases also scavenge NO, or diminish its bioavailability via reactive oxygen species (ROS) production, suggesting an intricate balance. Moreover, nitrite, whether produced endogenously, or derived from exogenous nitrite or nitrate administration (including dietary sources via the Nitrate-Nitrite-NO pathway) beneficially modulates many key cardiovascular pathological processes.

In this review, we highlight the landmark studies which revealed nitrite's function in biological systems, and inspect its evolving role in cardiovascular protection. Whilst these effects have mainly been ascribed to the activity of one or more nitrite reductases, we also discuss newly-identified mechanisms, including nitrite anhydration, the involvement of s-nitrosothiols, nitro-fatty acids, and direct nitrite normoxic signalling, involving modification of mitochondrial structure and function, and ROS production. This article is part of a Special Issue entitled "Redox Signalling in the Cardiovascular System". (c) 2014 Elsevier Ltd. All rights reserved

Omar, S. A. and Webb, A. J., Nitrite reduction and cardiovascular protection, Journal of Molecular and Cellular Cardiology, 2014, 73, 57-69.

Nitrite reduction and cardiovascular protection

Inorganic nitrite, a metabolite of endogenously produced nitric oxide (NO) from NO synthases (NOS), provides the largest endocrine source of directly bioavailable NO. The conversion of nitrite to NO occurs mainly through enzymatic reduction, mediated by a range of proteins, including haem-globins, molybdo-flavoproteins, mitochondrial proteins, cytochrome P450 enzymes, and NOS. Such nitrite reduction is particularly favoured under hypoxia, when endogenous formation of NO from NOS is impaired. Under normoxic conditions, the majority of these nitrite reductases also scavenge NO, or diminish its bioavailability via reactive oxygen species (ROS) production, suggesting an intricate balance. Moreover, nitrite, whether produced endogenously, or derived from exogenous nitrite or nitrate administration (including dietary sources via the Nitrate-Nitrite-NO pathway) beneficially modulates many key cardiovascular pathological processes.

In this review, we highlight the landmark studies which revealed nitrite's function in biological systems, and inspect its evolving role in cardiovascular protection. Whilst these effects have mainly been ascribed to the activity of one or more nitrite reductases, we also discuss newly-identified mechanisms, including nitrite anhydration, the involvement of s-nitrosothiols, nitro-fatty acids, and direct nitrite normoxic signalling, involving modification of mitochondrial structure and function, and ROS production. This article is part of a Special Issue entitled "Redox Signalling in the Cardiovascular System". (c) 2014 Elsevier Ltd. All rights reserved

Omar, S. A. and Webb, A. J., Nitrite reduction and cardiovascular protection, Journal of Molecular and Cellular Cardiology, 2014, 73, 57-69.

Nitrite reductase and nitric-oxide synthase activity of the mitochondrial molybdopterin enzymes mARC1 and mARC2

Background: Nitrite reduction pathways are critical for biological NO production under hypoxia.

Results: The mitochondrial enzyme mARC reduces nitrite to NO using cytochrome b(5) as electron donor.

Conclusion: mARC forms an electron transfer chain with NADH, cytochrome b(5), and cytochrome b(5) reductase to reduce nitrite to NO.

Significance: mARC proteins may constitute a new pathway for hypoxic NO production in vivo.

Mitochondrial amidoxime reducing component (mARC) proteins are molybdopterin-containing enzymes of unclear physiological function. Both human isoforms mARC-1 and mARC-2 are able to catalyze the reduction of nitrite when they are in the reduced form. Moreover, our results indicate that mARC can generate nitric oxide (NO) from nitrite when forming an electron transfer chain with NADH, cytochrome b(5), and NADH-dependent cytochrome b(5) reductase. The rate of NO formation increases almost 3-fold when pH was lowered from 7.5 to 6.5.

To determine if nitrite reduction is catalyzed by molybdenum in the active site of mARC-1, we mutated the putative active site cysteine residue (Cys-273), known to coordinate molybdenum binding. NO formation was abolished by the C273A mutation in mARC-1.

Supplementation of transformed Escherichia coli with tungsten facilitated the replacement of molybdenum in recombinant mARC-1 and abolished NO formation.

Therefore, we conclude that human mARC-1 and mARC-2 are capable of catalyzing reduction of nitrite to NO through reaction with its molybdenum cofactor.

Finally, expression of mARC-1 in HEK cells using a lentivirus vector was used to confirm cellular nitrite reduction to NO. A comparison of NO formation profiles between mARC and xanthine oxidase reveals similar K-cat and V-max values but more sustained NO formation from mARC, possibly because it is not vulnerable to autoinhibition via molybdenum desulfuration.

The reduction of nitrite by mARC in the mitochondria may represent a new signaling pathway for NADH-dependent hypoxic NO production

Sparacino-Watkins, C. E., Tejero, J., Sun, B., Gauthier, M. C., Thomas, J., Ragireddy, V., Merchant, B. A., Wang, J., Azarov, I., Basu, P., and Gladwin, M. T., Nitrite Reductase and Nitric-oxide Synthase Activity of the Mitochondrial Molybdopterin Enzymes mARC1 and mARC2 SO JOURNAL OF BIOLOGICAL CHEMISTRY, 2014, 289, 10345-10358.