Cytochrome nitrite reduction

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... 

It is remarkable that the oxidized states of the cytochromes cdi from P pantotrophus and P. aeruginosa have different structures. It is not clear at present whether one of these structures is superior for catalyzing nitrite reduction. Certainly in the P. pantotrophus enzyme the ligand switching at both ligand centers upon changing oxidation state is... 

The major product of nitrite reduction is NO (Denariaz et ai, Iwasaki and Matsubara, 1972 Kakutani et al., 1981 Liu et ai, 1986 Masuko et ai, 1984 Sawada et ai, 1978 Zumft et ai, 1987) just as is the case with the cytochrome cd, type enzyme. As discussed previously in this chapter, the Cu-type enzyme from A. cycloclastes has been reported to reduce nitrite to N2O in the presence of NO (Averill and Tiedje, 1990 Jackson et ai, 1991), a catalytic capability apparently not exhibited by heme-type nitrite reductases. 

Shoun, H., and Tanimoto, T. (1991). Denitrification by the fungus Fusarium oxysporum and involvement of cytochrome P-450 in the respiratory nitrite reduction. J. Biol. Chem. 266, 11078-11082. 

Heme containing proteins are also involved in nitrite reduction. Cytochrome... 

Yamanaka and co-workers (364-366) have crystallized a cytochrome oxidase from P. aeruginosa which oxidizes Pseudomonas ferrocytochrome c-551. It is also capable of nitrite reduction with a turnover number of 4000 moles nitrite reduced under anaerobic conditions to nitric oxide per minute at 37°. It is an adaptive enzyme, nitrate being essential for its biosynthesis. The enzyme has a molecular weight of 120,000, with two subunits of equivalent molecular weight, 2 heme c and 2 heme d groups per mole (Fig. 38) (366a). Nitrite reductase activity is 94% inhibited by 8 X 10 M KCN, but only by CO. The lack of CO inhibition appears to be related to the fact that the enzyme has a greater affinity for nitrite than for carbon monoxide. 

There are four different types of nitrite reductases the copper-containing protein Copper Enzymes in Denitrification and cytochrome cd perform a one-electron rednetion of nitrite to nitric oxide, and are involved in denitrification " the siroheme-containing protein and the cytochrome c ititrite reductase (cNiR) both perform the complete, six-electron reduction, of nitrite to ammonia. The cNiR is present in the y, 5 or e-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, ntfA. 

Cytochrome cd nitrite reductase from Paracoceus pantotrophus has a different mechanism, with two identical subunits, each with domains containing a c-type cytochrome heme and a dj-type cytochrome heme. Electrons from external donors enter through the c heme the d heme is the site of nitrite reduction to NO and oxygen reduction to water. One of the puzzles of the mechanism is how the NO can escape from... 

FIGURE 16-15 Possible Routes for Nitrite Reduction by Cytochrome cdj Nitrite Reductase. (Redrawn from G. Ranghino, E. Scorza, T. Sjogren, P. A. Williams, M. Ricci, and J. Hajdu, Biochemistry, 2000, 39, 10958.)... 

Direct reaction of NO with enzymes has been shown for cytochrome c oxidase (cyt c oxidase). The reaction of NO with the binuclear metal centre of cyt c oxidase apparently leads to the formation of nitrite at the active site [123] the mechanism of which was described as the opposite of nitrite reduction to NO by non-haem nitrite reductases [124]. The inhibition was caused by the binding of NO to the reduced copper centre of the enzyme rather than the expected reaction with Fe. ... 

Nitrite reduction to nitric oxide is catalyzed by dissimilatory nitrite reductase. The enzymes purified from Alcaligenes faecalis (Iwasaki and Matsub-ara, 1971), Pseudomonas aeruginosa (Walker and Nicholas, 1961), andMj-crococcus denitrificans (Newton, 1969) have been shown to contain c d-type cytochrome. The nitrite reductase from Achromobacter cycloclastes does not... 

The function of cytochromes in dissimilatory nitrite reduction does not appear to be one of electron transport. Payne et al. (1971) showed that epr studies indicated no metal involvement but there was a formation of a heme-nitric oxide complex during nitrite reductase action. 

Reaction mechanism Based on the observation of reaction intermediates in the crystal structure and on quantum chemical calculations Einsle et al. [148] propose an outline of the first detailed reaction mechanism of the cytochrome c Nir from W. succinogenes. Nitrite reduction starts with a het-erolytic cleavage of the weak N-O bond, which is facilitated by a pronounced backbonding interaction between nitrite and the reduced active site iron. The protons come firom a highly conserved histidine and tyrosine. Elimination of one of both amino acids results in a significant reduced activity. Subsequently, two rapid one-electron reductions lead to a FeNO form and, by protonation, to a HNO adduct. A further two-electron two-proton step leads to hydroxylamine. The iron in the hydroxylamine complex is in the Fe(III) state [149], which is unusual compared to synthetic iron-hydroxylamine complexes where the iron is mainly in the Fe(II) state. Finally, it readily loses water to give the product, ammonia. This presumably dissociates firom the Fe(III) form of the active site, whose re-reduction closes the reaction cycle. 

Heme di (107), which was isolated by Timkovich et al (70) and Chang et al (71) occurs as one of two cofactors in the reductase cytochrome cd. Cytochrome cd participates in the reduction of nitrite to nitrous oxide (N2O) in chemoautotrophic bacteria, such as Pseudomonas aeruginosa, Paracoccus denitrificans, and Thiobacillus denitrificans (13). From recent investigations it seems very likely that cytochrome cdi mediates the nitrite reduction to nitric oxide (NO) and that a second enzyme produces N2O from NO (13). A structure was... 

In the 1990s, Averill described the putative intermediate of the initial steps in nitrite reduction, a ferric nitrosyl, as characterized by FTIR" . Recent work has identified this ferric species, using time-resolved stop-flow experiments, that is in apparent equilibrium with a ferrous nitrosyl, previously thought to be a catalytic dead end . Both the ferric and ferrous nitrosyl adducts are relatively long-lived, and no study has thus far demonstrated catalytically competent NO release from cytochrome cd. Therefore, certain mechanistic aspects remain obscure. 

Lojou and coworkers have investigated catalytic nitrite reduction by cytochrome cd NiR using cyclic voltammetry For this enzyme, direct electrochemistry was not possible and superimposition of absorption spectra made spectroelectrochemical analysis difficult but catalysis could be driven... 

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... 

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