NADPH-nitrate reductase

Padidam, M., Venkatesvarlu, K. Johri, M.M. (1991). Ammonium represses NADPH-nitrate reductase in the moss Funaria hygrometrica. Plant Science 75, 184-94. 

The physiological roles of the phenolates produced by B. thuringien-sis are not known. Neither the slow band nor the intermediate band serves as the molybdenum cofactor in the in vitro restoration of NADPH-nitrate reductase. It is reasonable nevertheless to posulate that these compounds are excreted by this organism in nature and that under the appropriate environmental conditions, these phenolates will coordinate tungstate, ferric iron, or molybdate. 

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... 

NADPH DEHYDROGENASE NITRATE REDUCTASE PROTOPORPHYRINOGEN OXIDASE SARCOSINE DEHYDROGENASE SULFITE REDUCTASE FoFi-ATPase (and RELATED PROTONTRANSPORTING ATPases)... 

Similar mechanisms operate in the action of nitrate reductase and nitrite reductase. Both of these substances are produced from ammonia by oxidation. Plants and soil bacteria can reduce these compounds to provide ammonia for metabolism. The common agricultural fertilizer ammonium nitrate, NH4NO3, provides reduced nitrogen for plant growth directly, and by providing a substrate for nitrate reduction. NADH or NADPH is the electron donor for nitrate reductase, depending on the organism. 

Many species of bacteria also have an assimilatory nitrite reductase which is located in the cytoplasm. There is relatively little known about such enzymes but the electron donor is throught to be NADPH and the active site again has siroheme (Cole, 1988). The assimilatory nitrite reductases of both plants and bacteria use nitrite that is provided as the product of the assimilatory nitrate reductases. Nitrate is a very common natural N source for plant and bacterial growth. 

In all photoautotrophs, reduction of NOj" to NH4 is achieved in two distinct enzymatic steps (Campbell, 2001). First, assimilatory nitrate reductase (NR) catalyzes the two electron reduction from NOj" to NO2. NR is a large soluble cytoplasmic enzyme with FAD (flavin adinine dinucleotide), an iron-containing cytochrome and molybdopterin prosthetic groups, and requires NADH and/or NADPH as an electron donor (Guerrero et al, 1981). Functional NR is in the form of a homodimer and therefore requires two atoms of iron per enzyme. Following transport into the chloroplast, NO2 undergoes a 6 e reduction to NH4 via assimilatory nitrite reductase (NiR). NiR, a soluble chloroplastic enzyme, contains five iron atoms per active enzyme molecule, and requires photosynthetically reduced ferredoxin as an electron donor (Guerrero et al., 1981). 

This two-electron reduction uses NADH or NADPH as electron donor depending on the particular nitrate reductase (EC 1.7.1.1 or EC 1.7.1.2, respectively). Nitrite is then reduced to ammonium in a six-electron process that involves the transfer of three electron pairs from NAD(P)H ... 

The two-domain, structural motif in FNR represents a common structural feature in a large class of enzymes that catalyze electron transfer between a nicotinamide dinucleotide molecule and a one-electron carrier. Beside the photosynthetic electron-transfer enzyme, others non-photosynthetic ones include flavodoxin reductase, sulfite reductase, nitrate reductase, cytochrome reductase, and NADPH-cyto-chrome P450 reductase. FNR belongs to the group of so-called dehydrogenases-electron transferases, i.e., flavoproteins that catalyze electron transfer from two, one-electron donor molecules to a single two-electron acceptor molecule. 

The uptake of nitrate and subsequent conversion to reduced nitrogen in cells requires a change of five in the oxidation state and proceeds in a stepwise fashion. The initial reduction takes place via the nitrate/nitrite reductase enzyme present in phytoplankton and requires large amounts of the reduced nicotinamide-adenine dinucleotide phosphate (NADPH) and of adenosine triphosphate (ATP) and thus of harvested light energy from photosystem II. Both the nitrogenase enzyme and the nitrate reductase enzyme require iron as a cofactor and are thus sensitive to iron availability. 

Pyridine nucleotide mediated nitrate reductase and NADPH cytochrome c reductase activities in the purified preparations are inhibited by p-hydroxymercuribenzoate (Garrett and Nason, 1969 McDonald and Coddington, 1974 Guerrero and Gutierrez, 1977). This inhibition could be overcome with cysteine or dithiothreitol. Cyanide and azide inhibit pyridine nucleotide and reduced viologen dye mediated nitrate reduction but do not affect NADPH cytochrome c reductase activity. 

Studies with Neurospora crassa and Aspergillus niger indicate that with wild strains the presence of nitrate is required for synthesis of nitrate reductase with NADPH and reduced viologen dye activity. The producticm of aberrant nitrate reductase by mutant strains does not require nitrate, thus the enzyme is constitutive (Cove and Pateman, 1%9 Dantziget ai, 1978). 

In addition to the substrate, nitrate, regulating the production of nitrate reductase, it has been reported that in some instances the product nitrite can stimulate the appearance of nitrate reductase (Ingle et al., 1966 Kaplan cr al., 1978). The aromatic nitro compound, chloramphenicol, and other organic nitro compounds have been shown to regulate the level of nitrate reductase in rice seedling (Shen, 1972a,b). Not only is the level of nitrate reductase influenced by these compounds but the nucleotide specificity is changed in favor of NADPH. 

The single cell green alga Chlorella produces a nitrate reductase that has been investigated by Kay et al The natural reductant is NADPH and in the presence of NR and NADPH the reduced NR enzyme is able to donate electrons to cytochrome c as evident from the appearance of a catalytic cytochrome c oxidation wave. Reduced MV and diquat were effective artificial electron donors to NR and in the presence of nitrate a catalytic reduction current was observed. 

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