Cytochrome c nitrite reductase

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 . 

Cytochrome c nitrite reductase (bacteria) Reduces HA and NH2OCH3 to ammonia (NH3) 10, 23... 

Blom was the first to demonstrate, in 1928, the formation of HA by an unknown mixture of bacteria which utilized nitrate as their sole nitrogen source to produce ammonia , an observation substantiated by Lindsey and Rhines who generalized this reaction to a diverse set of microorganisms capable of producing NH3 by reduction of both nitrites and nitrates. The mechanism of the 6-electron reduction of nitrite to ammonia (i.e. conversion of the [N + 02] species to by bacterial cytochrome c nitrite reductase... 

Desulfovibrio desulfuricans Cytochrome C nitrite reductase lOAH 1 HisL 62 AspL His cr... 

Fe (heme) Hemoglobin, peroxidase, catalase, cytochrome P-450, tryptophan dioxygenase, cytochrome c, nitrite reductase... 

D.J., and Butt, J.N. (2002) Protein film voltammetry reveals distinctive fingerprints of nitrite and hydroxylamine reduction by a cytochrome c nitrite reductase. Journal of Biological Chemistry, 277, 23374-23381. 

Unfortunately, most of the M-HNO complexes are insoluble in water, in contrast with MbnHNO, which shows to be remarkably inert toward the release of UNO (hour timescale). The latter fact supports the proposal of UNO being a long-lived intermediate in the disproportionation reaction of NH2OH catalyzed by pentacyanofer-rates, leading to bound NO+ as the final oxidized product,67 or in the six-electron reduction of SNP with 1,4-dimethylhydrazine (Equation 7.15).42 On the basis of crystallographic observation of some intermediates and DFT calculations, UNO has been proposed as a necessary intermediate in the six-electron reduction of N02 to NH3 catalyzed by cytochrome c nitrite reductase.68... 

There are four different types of nitrite reductases the copper-containing protein Copper Enzymes in Denitrification and cytochrome cd perform a one-electron reduction of nitrite to nitric oxide, and are involved in denitrification the siroheme-containing protein and the cytochrome c nitrite reductase (cNiR) both perform the complete, six-electron reduction, of nitrite to ammonia. The cNiR is present in the y, 5 or -subclasses of proteobacteria, and is encoded by the nrf operon (nitrite reduction with /ormate), which has different gene composition in the different classes of bacteria, having in common only the gene for the catalytic subunit, nrfA. 

Einsle, in Cytochrome c Nitrite Reductase, in Handbook of Metaiioproteins , eds. A. Messerschmidt, R. Huber, T. Pouios, and K. Wieghardt, John Wiley Sons, New York, 2001, p. 440. 

Nitroxyl (HNO/NO ) heme-model complexes ( FeNO , according to the Enemark-Feltham notation) have received special attention due to the intermediacy of nitroxyl-heme adducts in a variety of catalytic processes related to the biogeochemical cycle of nitrogen (104). For example, for the six-electron reduction of nitrite to ammonia that is catalvzed by cytochrome c nitrite reductase (ccNir), a heme FeNO complex is proposed as an intermediate (Scheme 5) (105,106). This intermediate has also been suggested for the reduction of NO to N2O by P450nor (Scheme 6) (107). Then, the isolation of a suitable FeNO heme complex that allows structural and functional characterizations will help to imderstand the reaction mechanism of ccNir and other enz5mies. 

The reduction of nitrite to ammonia was described for bacteria with a fermentative rather than a respiratory metabolism [125]. However, growth of various bacteria by oxidation of non-fermentable substrates such as formate linked to the reduction of nitrite to ammonia, demonstrated that nitrite ammonification may also function as respiratory energy conserving process [126]. The enzymol-ogy and bioenergetics of respiratory nitrite ammonification have been recently reviewed [17]. In respiratory nitrite ammonification, nitrite is reduced to ammonia without the release of intermediate products, such as NO or N2O, in a six-electron step by a cytochrome c nitrite reductase, the so-caUed NrfA protein [Eq. (10)] [127-131]. 

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 . 

Gwyer, J.D., H.C. Angove, D.J. Richardson, and J.N. Butt (2004). Redox-triggered events in cytochrome c nitrite reductase. Bioelectrochemistry 63, 43-47. 

Rudolf, M., O. Einsle, F. Neese, and P.M.H. Kroneck (2002). Pentahaem cytochrome c nitrite reductase Reaction with hydroxylamine, a potential reaction intermediate and substrate. Biochem. Soc. Transact. 30, 649-653. 

Two major pathways have been shown to exist in nitrite reduction [274]. In the first pathway, nitrite is reduced to NO, while in the second there is a direct conversion of nitrite to NH3 or NH4" ". Two classes of nitrite reductase (NIR), namely the cytochromes cd [274], and the copper nitrite reductase [274], have been identified for the first pathway and two classes of enzyme, namely the siroheme nitrite reductase and cytochrome c nitrite reductase, have been proposed to follow the second pathway. The mechanism of these four enzymes has been recently reviewed [274], and only a brief summary of the electron-transfer reactions of cytochrome cd nitrite reductase will be given here. The initial step in the conversion of NO2 to NO involves a binding of the nitrite ion to the metal of the reduced heme. This first step is followed by the uptake of two protons and the loss of one water molecule to yield an electrophilic ferrous pe +-NO+ species, also formulated as a pe +-NO" complex. The dissociation of NO from this species produces the ferric heme d, which is in turn reduced back to its original state by heme c. Why the eri2yme does not reduce the nitrosyl species, Fe -]s[0 or Fe -NO to its Fe -NO form, prior to dissociation of NO in the heme, has been discussed in the literature [274], and may... 

NO2 Cytochrome C nitrite reductase in sol-gel on pyrol5dic graphite electrode Bio-catalysis [212]... 

In cytochrome c nitrite reductase, a PCET mechanism was established using a combination of catalytic and non-catalytic PFV. ° ... 

Almeida MG, Silveira CM, Guigliaielli B, Bertrand P, Moura JJG, Moura I, Leger C (2007) A needle in a haystack the active site of the membrane-bound complex cytochrome c nitrite reductase. FEBS Lett 581 284—288... 

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