Nitrite reductases assimilatory

4Fe-4S cluster. An identical active site is found in the siroheme sulfite reductases (SiR) that catalyze the six-electron reduction of SOj to H2S. The similarity of the enzymes extends to their activity, SiR will catalyze nitrite reduction and NiR will catalyze sulfite reduction the main difference being a greater affinity (by several orders of magnitude) for each enzyme s specific substrate . 

A very similar catalytic cycle has been proposed for the cytochrome c nitrite reductases, ccNiR, which serve in some anaerobes as terminal electron acceptors . The ccNiR has five type c hemes per monomer and a lysine-coordinated heme at the active site. Potentiometric titrations showed enzyme s unique lysine-coordinated heme, labeled heme 1, has an Fe reduction potential at —107 mV. The other hemes with bis-histidine coordination, termed hemes 2 through 5, have reduction potentials between -37 and —323 mV. Tentative assignments of each heme s potentials have been proposed using EPR titration data correlated with a recently determined structure . 

Under nitrite-limited conditions, an increased catalytic activity occurred at -105 mV, close to the reduction potentials of heme 1 and 3 ca. -107 mV) and decreased ca. -320 mV, possibly due to a conformational change caused by reduction of hemes 4 and/or 5 ca. -323 mV), or inhibition due to ferrous nitrosyl formation . As the concentration of nitrite increased, the catalytic peak broadened and moved toward a lower potential under these enzyme-limited conditions. 

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In order to check whether the occurrence of the Rieske-type sequence motif is unique for the assimilatory nitrite reductase from Bacillus subtilis, the sequences of other assimilatory nitrite reductases were searched for the presence of the four putative ligands of Rieske-type clusters. A well-conserved sequence pattern... 

Vega, J. M., Garrett, R. H., and Siegel, L. M. (1975). Siroheme A prosthetic group of the Neurospora crassa assimilatory nitrite reductases. ]. Biol. Chem. 250, 7980-7989. 

Assimilatory nitrite reductases of plants, fungi, and bacteria carry out the six-electron reduction of nitrite to ammonium ions (Eq. 24-13) using electron donors such as reduced ferredoxins or NADPH. 

Green plants, algae, fungi, cyanobacteria and bacteria that assimilate nitrate also produce assimilatory nitrite reductases, which catalyze the six-electron reduction of nitrite to ammonia (equation 89). The formation of heme-nitrosyl intermediates has been detected in several cases,1515 while hydroxylamine is commonly thought to be an intermediate. Added hydroxylamine is rapidly reduced to ammonia. However, no intermediates are released, and ammonia is the only product... 

However, in both the yeast and the E. coli systems, the stoichiometries for NADPH S and SOj -rS - are 3 1 and 1 1, respectively. These results and the inability to detect 2-electron- and 4-electron-reduced intermediates in these systems have suggested that such intermediates, if present at all, must be firmly held on the surface of the enzyme. It has further been suggested that the presence of multiple flavins and hemes in the enzyme might be a device for achieving a rapid six-electron reduction of sulfite without the release of intermediates (414)- This situation is analogous to the four-electron reduction of 0, to 2H,0 by cytochrome oxidase and the six-electron reduction of nitrite to ammonia by various assimilatory nitrite reductases. However, unlike cytochrome oxidase,... 

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 various species of bacteria several different types of non-assimilatory nitrite reductases are found. Escherichia coli has a cytoplasmic NAD(P)H-dependent enzyme whose role seems to be detoxification of nitrite. This type of enzyme, coded for by the nirB gene, also contains siroheme as the redox active catalytic center (Cole, 1988). Additionally in E. coli, and expressed under different conditions to the cytoplasmic enzyme, is a periplasmic nitrite reductase that catalyses formation of ammonia from nitrite (Cole, 1988). This enzyme has five c-type (Figure 1) hemes per polypeptide chain one of these hemes, the catalytic site, has the unique CXXCK sequence as its attachment site (Einsle et al., 1999). Electrons reach this type of nitrite reductase, which is fairly widely distributed amongst the microbial world, from the cytoplasmic membrane electron transfer chain. The exact electron donor partner from such chains for this type of nitrite reductase is unknown (Berks et al., 1995). 

As potential models for the assimilatory nitrite reductases, electrochemical reduction of nitrosyliron(II) porphyrins (see Nitrosyl Complexes), PFeNO, yields first the Fe -NO complex, followed by two additional one-electron reductions. In the presence of phenols, the product of electrolysis is hydroxylamiue. " ... 

In addition, there have been several reports of reduced porphyrin or oxophlorin tt-cation radicals of Fe. Since it has been suggested that Fe isobacteriochlorin TT-cation radicals are viable species for at least two reaction states of the hemoprotein subunit of E. coli sulfite reductase and assimilatory nitrite reductases from several organisms, and at least one state of the heme d (chlorin) center of the dissimilatory nitrite reductases from several microorganisms, the investigation of the one-electron reduced states of these proposed intermediates has also been undertaken. 

Figure 12 A diagram of the nitrogen cycle with catalyzing enzymes and metal requirements of each step. NIT, nitrogenase AMO, ammonium mono-oxygenase HAO, hydroxylamine oxidoreductase NAR, membrane-bound respiratory nitrate reductase NAP, periplasmic respiratory nitrate reductase NR, assimila-tory nitrate reductase NIR, respiratory nitrite reductase NiR, assimilatory nitrite reductase NOR, nitric oxide reductase N2OR, nitrous oxide reductase.

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). 

Lafferty MA and Caret RH (1974). Purification and properties of the neurospora crassa assimilatory nitrite reductase. J Biol Chem, 249, 7555-7557. 

Two distinct reactivities are observed assimilatory nitrite reductases... 

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