Nitrite reductase cytochrome

Nitrite reductase (NAD(P)H) [EC 1.6.6.4] catalyzes the reaction of three NAD(P)H with nitrite to yield three NAD(P)+, NH4OH, and water. Cofactors for this enzyme include FAD, non-heme iron, and siroheme. (2) Nitrite reductase (cytochrome) [EC 1.7.2.1] is a copper-depen-dent system that catalyzes the reaction of nitric oxide with two ferricytochrome c and water to produce nitrite and two ferrocytochrome c. (3) Ferredoxin-nitrite reductase [EC 1.7.7.1], a heme- and iron-dependent enzyme, catalyzes the reaction of ammonia with three oxidized ferredoxin to produce nitrite and three reduced ferredoxin. (4) Nitrite reductase [EC 1.7.99.3] is a copper- and FAD-dependent enzyme that catalyzes the reaction of two nitric oxide with an acceptor substrate and two water to produce two nitrite and the reduced acceptor. 

The dissimilatory nitrite reductase (cytochrome cdlt see below) catalyzes the nitrosation of several nucleophiles by nitrite.1531 This may occur through a heme-nitrosyl in which an NO+ group is present. Coordinated nitrite in simple metal complexes such as [Run(NH3)5(N02)]+... 

No similar tryptophan docking motifs are present in the 7-bladed superbarrel proteins galactose oxidase and methylamine dehydrogenase, or in the 8-bladed nitrite reductase (cytochrome cJi) (Fulop et a/., 1995 Baker et al., 1997). 

Cheesman, M., Ferguson, S. J., Moir, J. W. B., Richardson, D. J., Zumft, W. G., and Thomson, A. J., 1997, Two enzymes with a common function but different heme ligands. The optical and magnetic properties of the heme groups in the oxidised forms of nitrite reductase, cytochrome cdi, from Pseudomonas stutzeri and Thiosphaera pantotropha. Biochemistry 36 16267916276. 

Still another nitrite reductase, cytochrome c NIR, contains five heme groups, only one of which functions as the active site. A combination of calculations and crystallographic studies has suggested a mechanism in which nitrite replaces a water molecule on one side of the Fe(II) heme (a lysine N is on the opposite side), one of the oxygens of N02 is protonated, and the N — 0 bond is broken with loss of H2O, leaving a linear Fe(III)—NO species with a low-spin Fe(III). Addition of two electrons and H" " leads to Fe HNO, which is then reduced to Fe H2NOH. Yet another electron and another allow release of H2O and formation of an Fe NH3 complex. Release of ammonia and a final electron and water addition complete the cycle. Overall, six electrons and seven hydrogen ions react with the nitrite ... 

Direct electrochemistry has also been used (72-78) to couple the electrode reactions to enzymes for which the redox proteins act as cofactors. In the studies, the chemically reduced or oxidized enzyme was turned over through the use of a protein and its electrode reaction as the source or sink of electrons. In the first report (72, 73) of such application, the electrochemical reduction of horse heart cjd,ochrome c was coupled to the reduction of dioxygen in the presence of Pseudomonas aeruginosa nitrite reductase/cytochrome oxidase via the redox proteins, azurin and cytochrome C551. The system corresponded to an oxygen electrode in which the four-electron reduction of dioxygen was achieved relatively fast at pH 7. 

Heme d,6 another isobacteriochlorin, occurs as one of two cofactors in the reductase cytochrome cdj which mediates the nitrite reduction to nitrogen monoxide (NO) and from there to dinitrogen oxide (N20) in denitrifying bacteria.7... 

Fe Cytochrome oxidase reduction of oxygen to water Cytochrome P-450 0-insertion from O2, and detoxification Cytochromes b and c electron transport in respiration and photosynthesis Cytochrome f photosynthetic electron transport Ferredoxin electron transport in photosynthesis and nitrogen fixation Iron-sulfur proteins electron transport in respiration and photosynthesis Nitrate and nitrite reductases reduction to ammonium... 

STRUCTURE AND ENZYMOLOGY OF TWO BACTERIAL DIHEME ENZYMES CYTOCHROME cd NITRITE REDUCTASE AND CYTOCHROME c PEROXIDASE... 

Fe atoms. It had been anticipated that the c-type cytochrome center would have His/Met coordination, but His/His is observed. The former is the more usual coordination, especially at the high potential end E° > +200 mV) ofthe typical bacterial electron transfer chain to which the nitrite reductase is connected (Fig. 2) (7). The second curious feature is that the di heme iron is also six-coordinate thus, the enzyme does not offer a substrate-binding site at either heme. In addition to an expected axial histidine ligand there was an axial tyrosine (residue 25) ligand to the d heme (Fig. 4a). Each monomer is organized into two domains. 

Such an involvement of an amino acid side-chain ligand switch within each catalytic cycle was a novel proposal and as such needs to be scrutinized by a variety of experimental procedures as well as analysis in the context of information known for cytochrome cd nitrite reductase from another source (see later discussion). However, it is interesting to note that something similar has been proposed for the protocate-chuate 3,4-dioxygenase enzyme from Pseudomonas putida (15). On the other hand, bacterial cytochrome c peroxidase offers an example where ligand switching seemingly relates only to an activation phenomenon. 

It has long been assumed that azurin is an in vivo electron donor to cytochrome cdi of P. aeruginosa. The construction of mutants of P. aeruginosa in which one or both of the genes for azurin and cytochrome C551 have been deleted has led to the conclusion that in vivo cytochrome C551 is essential for the donation of electrons to the nitrite reductase and that azurin is ineffective (24). The discrepancy between in vivo and in vitro observations either could be reconciled if it is the failure of azurin to accept electrons from the cytochrome bc complex or another donor that is responsible for its ineffectiveness in vivo. 

Since HA is unstable in vivo , and is known to rapidly associate with the heme part of heme proteins , and possibly also with a variety of biological oxidants, such as the superoxide anion that is produced by many mammalian cells, it is difficult to demonstrate its accumulation in vivo. Already in 1932 Lindsey and Rhines discussed some analytical difficulties in the detection of HA, since when added externally, it disappeared rapidly from bacterial cultures this led to the conclusion that even if it is produced as an intermediate, its consumption is too fast to allow the accumulation of sufficient quantities for analytical demonstration. Compelling indirect evidence for the presence of HA as an intermediate in the enzymatically catalyzed reduction of nitrite (N02 ) to NH3 was provided by Einsle and colleagues , who characterized the crystal structure of the complex obtained by soaking cytochrome c-nitrite reductase with NH20H. ... 

The involvement of HA during bacterial conversion of nitrate to NH3 (known also as the nitrate ammonification phase of the nitrogen cycle) has been studied at the molecular level as part of an effort to delineate the mechanism of conversion of nitrite to NH3 by a group of multiheme cytochromes of bacterial origin. The overall reduction reaction is depicted in equation 3 for cytochrome c-nitrite reductase " ... 

Based on crystallographic observations it was suggested that the HA intermediate is bound to the cytochrome reductase via the iron atom, Fe(II)—NH2OH, and undergoes subsequent reduction to produce the NH3 that then dissociates from the protein ". It is of interest that the specific activity of cytochrome c-nitrite reductase from S. deleyianum in the conversion of N02 to NH3 is only 2-fold greater than that recorded for the conversion of HA to ammonia by the same enzyme, an observation that strongly supports the involvement of HA as an intermediate in the catalytic reduction of nitrite to NH3 . 

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