Nitrate reductase molybdenum

Molybdenum. Molybdenum is a component of the metaHoen2ymes xanthine oxidase, aldehyde oxidase, and sulfite oxidase in mammals (130). Two other molybdenum metaHoen2ymes present in nitrifying bacteria have been characteri2ed nitrogenase and nitrate reductase (131). The molybdenum in the oxidases, is involved in redox reactions. The heme iron in sulfite oxidase also is involved in electron transfer (132). 

The element molybdenum (atomic weight 95.95) constitutes 0.08% of the weight of nitrate reductase. If the molecular weight of nitrate reductase is 240,000, what is its likely quaternary structure ... 

Sulfate reducers can use a wide range of terminal electron acceptors, and sulfate can be replaced by nitrate as a respiratory substrate. Molybdenum-containing enzymes have been discovered in SRB (also see later discussion) and, in particular, D. desulfuricans, grown in the presence of nitrate, generates a complex enzymatic system containing the following molybdenum enzymes (a) aldehyde oxidoreduc-tase (AOR), which reduces adehydes to carboxylic acids (b) formate dehydrogenase (FDH), which oxidizes formate to CO2 and (c) nitrate reductase (the first isolated from a SRB), which completes the enzy-... 

The molyhdopterin cofactor, as found in different enzymes, may be present either as the nucleoside monophosphate or in the dinucleotide form. In some cases the molybdenum atom binds one single cofactor molecule, while in others, two pterin cofactors coordinate the metal. Molyhdopterin cytosine dinucleotide (MCD) is found in AORs from sulfate reducers, and molyhdopterin adenine dinucleotide and molyb-dopterin hypoxanthine dinucleotide were reported for other enzymes (205). The first structural evidence for binding of the dithiolene group of the pterin tricyclic system to molybdenum was shown for the AOR from Pyrococcus furiosus and D. gigas (199). In the latter, one molyb-dopterin cytosine dinucleotide (MCD) is used for molybdenum ligation. Two molecules of MGD are present in the formate dehydrogenase and nitrate reductase. 

The molybdenum cofactor was liberated from D. gigas AOR, and under appropriate conditions was transferred quantitatively to nitrate reductase in extracts of Neurospora crassa nit-1 mutant) to yield active nitrate reductase 217). On the basis of molybdenum content, the activity observed for reconstitution with molybdenum cofactor of D. gigas was lower (25%) than the values observed for the procedure using extractable molybdenum cofactor of XO, used as reference. This result can now be put in the context of the difference in pterin present (MPT-XO and MCD-AOR) 218). 

D. desulfuricans is able to grow on nitrate, inducing two enzymes that responsible for the steps of conversion of nitrate to nitrite (nitrate reductase-NAP), which is an iron-sulfur Mo-containing enzyme, and that for conversion of nitrite to ammonia (nitrite reduc-tase-NIR), which is a heme-containing enzyme. Nitrate reductase from D. desulfuricans is the only characterized enzyme isolated from a sulfate reducer that has this function. The enzyme is a monomer of 74 kDa and contains two MGD bound to a molybdenum and one [4Fe-4S] center (228, 229) in a single polypeptide chain of 753 amino acids. FXAFS data on the native nitrate reductase show that besides the two pterins coordinated to the molybdenum, there is a cysteine and a nonsulfur ligand, probably a Mo-OH (G. N. George, personal communication). 

FPR studies at low temperature detect the presence of one iron-sulfur center and molybdenum. At low temperature a sample of nitrate reductase reduced by dithionite shows a rhombic signal (gm,x = 2.04, gmed = 1.94, and gnm = 1.90). This signal accounts for 0.84 spins/... 

The chlorate reductase has been characterized in strain GR-1 where it was found in the periplasm, is oxygen-sensitive, and contains molybdenum, and both [3Fe-4S] and [4Fe-4S] clusters (Kengen et al. 1999). The arsenate reductase from Chrysiogenes arsenatis contains Mo, Fe, and acid-labile S (Krafft and Macy 1998), and the reductase from Thauera selenatis that is specific for selenate, is located in the periplasmic space, and contains Mo, Fe, acid-labile S, and cytochrome b (Schroeder et al. 1997). In contrast, the membrane-bound selenate reductase from Enterobacter cloacae SLDla-1 that cannot function as an electron acceptor under anaerobic conditions contains Mo and Fe and is distinct from nitrate reductase (Ridley et al. 2006). 

For nitrate reductase, evidence on the role of molybdenum in the catalytic mechanism of the enzyme from Neurospora was first presented in 1954 by Nicholas and Nason (21) and the position seems to have changed relatively little since then. The original conclusion (23) was that molybdenum functions as an electron carrier in the sequence ... 

So little is known about molybdenum enzymes other than milk xanthine oxidase that there is little to be said by way of general conclusions. In all cases where there is direct evidence (except possibly for xanthine dehydrogenase from Micrococcus lactilyticus) it seems that molybdenum in the enzymes does have a redox function in catalysis. For the xanthine oxidases and dehydrogenases and for aldehyde oxidase, the metal is concerned in interaction of the enzymes with reducing substrates. However, for nitrate reductase it is apparently in interaction with the oxidizing substrate that the metal is involved. In nitrogenase the role of molybdenum is still quite uncertain. 

Kroneck PMH, Aht DJ (2002) Molybdenum in nitrate reductase and nitrite oxidoreductase. In Molybdenum and Tungsten- Their Roles in Biological Processes. Sigel A, Sigel H (eds) Marcel Dekker, Inc., New York, 369-403... 

Nitrate reductase (NADH) [EC 1.6.6.1], also known as assimilatory nitrate reduetase, eatalyzes the reaction of NADH with nitrate to produee NAD+, nitrite, and water. This enzyme uses FAD or FMN, heme, and a molybdenum ion as eofaetors. (2) Nitrate reductase (NAD(P)H) [EC 1.6.6.2], also known as assimilatory nitrate reduetase, eatalyzes the reaetion of NAD(P)H with nitrate to produee NAD(P)+, nitrite, and water. This enzyme uses FAD or FMN, heme, and a molybdenum ion as eofaetors. (3) Nitrate reductase (NADPH) [EC 1.6.6.3] eatalyzes the reaetion of NADPH with nitrate to produee NADP+, nitrite, and water. This enzyme uses FAD, heme, and a molybdenum ion as cofactors. (4) Nitrate reduetase (eytoehrome) [EC 1.9.6.1] catalyzes the reaetion of nitrate with ferroeytochrome to produce nitrite and ferrieytoehrome. (5) Nitrate reductase (ac-eeptor) [EC 1.7.99.4], also known as respiratory nitrate... 

METHOD OF CONTINUOUS VARIATION MOLYBDENUM COFACTOR (MoCo) Molybdenum-dependent reactions, ALDEHYDE OXIDASE MOLYBDOPTERIN NITRATE REDUCTASE NITROGENASE SULFITE OXIDASE XANTHINE DEHYDROGENASE MOLYBDOPTERIN... 

The opposite sequence, reduction of nitrate and nitrite ions, provides a major route of acquisition of ammonia for incorporation into cells by bacteria, fungi, and green plants (Fig. 24-1). Assimilatory (biosynthetic) nitrate reductases catalyze the two-electron reduction of nitrate to nitrite (Eq. 16-61). This is thought to occur at the molybdenum atom of the large 900-residue highly regulated793 molybdopterin-dependent enzyme. In green plants the reductant is... 

Bacterial assimilatory nitrate reductases have similar properties.86/86a In addition, many bacteria, including E. coli, are able to use nitrate ions as an oxidant for nitrate respiration under anaerobic conditions (Chapter 18). Tire dissimilatory nitrate reductases involved also contain molybdenum as well as Fe-S centers.85 Tire E. coli enzyme receives electrons from reduced quinones in the plasma membrane, passing them through cytochrome b, Fe-S centers, and molybdopterin to nitrate. The three-subunit aPy enzyme contains cytochrome b in one subunit, an Fe3S4 center as well as three Fe4S4 clusters in another, and the molybdenum cofactor in the third.87 Nitrate reduction to nitrite is also on the pathway of denitrification, which can lead to release of nitrogen as NO, NzO, and N2 by the action of dissimi-latory nitrite reductases. These enzymes873 have been discussed in Chapters 16 and 18. 

A molybdenum cofactor has been isolated from Proteus mirabilis 047, and has a molecular weight greater than 1000. The molybdoenzymes of E. coli, in addition to the formate dehydrogenases described above and the nitrate reductase (Section 62.1.9.6), also include the membrane-bound trimethylamine oxidase1044 and the soluble biotin sulfoxide reductase.1045... 

Nitrate reductases have been isolated from bacteria, plants and fungi and always contain molybdenum. Two types may be distinguished (a) the assimilatory nitrate reductases which catalyze the reduction of nitrate to nitrite, which ultimately is reduced to ammonia and used by... 

Nitrate reductase from Chlorella, an assimilatory enzyme, is a homotetramer of molecular weight 360 000 and contains one each of Mo, heme and FAD per subunit. The nitrate reductase from E. coli is a dissimilatory enzyme. EXAFS data are available on the molybdenum sites in both enzymes (Table 24).1050 The environment of the molybdenum in the assimilatory enzyme is similar to that found for sulfite oxidase, with at least two sulfur ligands near the molybdenum and a shuttle between monoxo and dioxo forms with redox change in the enzyme. This allows a similar mechanism to be put forward for the assimilatory nitrate reductase,1051 shown in equation (57), where an oxo group is transferred from nitrate to MoIV with production of nitrite and MoVI. 

The assimilatory nitrate reductase from Chlorella contains the molybdenum cofactor, as evidenced by the ability of the enzyme to donate the cofactor to the nitrate reductase of the mutant nit-1 of N. crassa. Reduction of the enzyme with NADH gives the Mov ESR signal, which is abolished on reoxidation with nitrate. Line shape and g values of the signal show a pH dependence similar to those observed previously for sulfite oxidase. The signal observed at pH 7.0 shows evidence for interaction with a single exchangeable proton.1053... 

An organism such as E. coli has at least five molybdoenzymes. It will be of great interest to look at the synthesis, availability and cellular distribution of the molybdenum cofactor and its relationship to the function of these molybdoenzymes at different stages of the cell cycle. The study of chlorate-resistant1057 and nitrate reductase-deficient1058 mutants of E. coli, which are... 

---