Reductases nitrate

Nitrate Reductase.—Reduction of nitrate by molybdenum(v) complexes of the type [M0OCI3L] and [MoOClL 2l where L and L are neutral or anionic bidentate ligands, proceeds by rate-determining loss of Cl in DMF at 25 °C  

Rate constants, ki, for L=phen and bipy are 4.45 x 10 s and 2.39 x 10 s respectively and subsequent steps involve inner-sphere co-ordination of nitrate and one-electron transfer. This is suggested as a model for nitrate reductase with disproportionation of the NO2 produced as the final step  

These complexes have unusual e.p.r. properties which have hitherto been considered to indicate sulphur co-ordination in model compounds and in flavo-proteins.  

1 Nitrate Reductase. - Nitrate reductase is a molybdenum-containing 

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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). 

Erythrityl is readily absorbed from the GI tract. It undergoes extensive first-pass metaboHsm ia the Hver by glutathione organic nitrate reductase. Time to onset of effect is 5—10 min by subHngual adrninistration and 20—30 min when swallowed. The duration of effects for the two routes ate up to 3 and 6 h, respectively. Adverse effects are similar to those described for nitroglycerin (99). 

Hemoprroteins contain heme Hemoglobin Cytochrome e Catalase Nitrate reductase Ammonium oxidase ... 

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 ... 

It is not clear why some organisms have two 14-3-3 isoforms while others have up to 12. Binding 14-3-3 inhibits the plant enzyme nitrate reductase and there appears to be no selectivity between plant 14-3-3 isoforms in fact yeast and human isoforms appear to work equally as well in vitro. The best example where selectivity has been demonstrated is human 14-3-3o. 14-3-3o Preferential homodimerizes with itself and crystallization revealed a structural basis for this isoform s dimerization properties as well as for its specific selectivity for target binding proteins. Here partner specificity is the result of amino acid differences outside of the phosphopeptide-binding cleft. 

Chlorella vulgaris nitrate reductase structure, 3,1438 Chlorides... 

Mariotti, A., Mariotti, F., Champigny, M.L., Amarger, N. and Moyse, A. 1982 Nitrogen isotope fractionation associated with nitrate reductase activity and uptake of NO3by pearl millet. Plant Physiology 69 880-884. 

Nitrate reductase (respiratory) (QH2 nitrate reductase) Glutamate synthase Aerobic bacteria NO3- + QH, NOf + q S[Fe,S,r [FesS ] " Mo-cofactor +GO 24,55... 

Fig. 6. Comparison of VTMCD spectra for biological [Fe3S4] clusters. (A) D. gigas Fdll (20) (B) P. furiosus 3Fe Fd (42) (C) A. vinelandii Fdl (70) (D) T. thermophilus 7Fe Fd (70) (E) E. coli nitrate reductase (24) (F) E. coli fumarate reductase (53) (G) spinach glutEimate synthase (25) (H) beef heart aconitase (27). Spectra were recorded at temperatures between 1.5 and 70 K with an apphed magnetic field of 4.5 T (sdl trEmsitions increase in intensity with decreasing temperature). BEmds originating from minor heme contaminEmts Eire indicated by an asterisk.

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/... 

There are four different classes of nitrate reductases (234). The nitrate reductases from D. desulfuricans show a strong homology to the a-subunit of the class of periplasmic respiratory nitrate reductases, and also to some of the enzymes that are included on the class of cytoplasmic assimilatory nitrate reductases. Because of this fact, a proposal was made for a new class of monomeric NAP, which contains the minimal arrangement of metal centers to perform nitrate reduction one [4Fe-4S] cluster and a Mo bound to two MGD. 

Fig. 6. Representative EPR spectra displayed by trinuclear and tetranucleEir iron-sulfur centers, (a) and (b) [3Fe-4S] + center in the NarH subunit of Escherichia coli nitrate reductase and the Ni-Fe hydrogenase fromD. gigas, respectively, (c) [4Fe-4S] + center in D. desulfuricans Norway ferredoxin I. (d) [4Fe-4S] center in Thiobacillus ferrooxidans ferredoxin. Experimental conditions temperature, 15 K microwave frequency, 9.330 GHz microwave power, (a) 100 mW, (b) 0.04 mW, (c) smd (d) 0.5 mW modulation amplitude (a), (c), (d) 0.5 mT, (b) 0.1 mT.

Similar difficulties have been encountered in the case of complex enzymes such as fumarate reductase and nitrate reductase from E. coli, in which substituting certain Cys ligands led to the loss of several if not all the iron-sulfur centers (171, 172). However, in the case of nitrate reductase, which possesses one [3Fe-4S] and three [4Fe-4S] centers, it was possible to remove selectively one [4Fe-4S]... 

Although the second Cys of the three groups of cysteine residues expected to coordinate the [4Fe-4S] centers of E. coli nitrate reductase were systematically mutated to induce their conversion into [3Fe-4S] centers, this conversion was achieved only in the case of the C247D mutation affecting the lowest potential [4Fe-4S] center (E. Valay, B. Guigliarelli, M. Asso, P. Bertrand, and F. Blasco, unpublished results, 1998). This result, which confirms the proposed coordination scheme (174), shows once again that the coordination capacity of the various Cys motifs of a protein can be differently affected by amino acid substitutions. 

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