Nitrite reductases denitrifying

Nitrite reductases (NiRs)—enzymes found in several strains of denitrifying bacteria— catalyze the one-electron reduction of nitrite anion to nitric oxide (Equation 1). - In addition to the importance of this process in the global nitrogen cycle (Figure 1), further incentive for the study of the denitrification process is provided by its environmental impact, ranging from the production of NO as a pollutant and NjO as a potent greenhouse gas, to lake eutrophication due to farm runoff that contains high concentrations of nitrates and nitrites. 

Nitrite reductase Denitrification Denitrifying bacteria Liu etal. (1986)... 

O-exchange studies of Ye et al. (1991) support, we believe, the catalysis by nitrite reductase of redox reversibility between nitrite and NO as depicted in the first line of Eq. (3). They observed by analyzing the 0 content of product N2O that all eight strains of denitrifying bacteria studied could catalyze the exchange of 0 between water and nitrite or NO by way of an electrophilic (nitrosyl donor) species of NO. The rates and extent of these exchange reactions depended on whether the bacterium made use of a heme- or Cu-type nitrite reductase. Contrary to the conclusions of Ye et al. (1991), we do not believe that this study otherwise informs about the pathway of denitrification or whether NO is an intermediate. 

Although the nitrite reductases of denitrifying bacteria seem to be NO-pro-ducing reductases in vitro, we cannot so easily say that they similarly are NO-producing in vivo. It is not so rare for an enzyme to catalyze more than one reaction or be served by more than one substrate, but it is virtually without precedence for an enzyme to change its chemistry with the same substrate between in vivo and in vitro situations. The reduction of nitrite in vivo has been probed by several means which target the activity of either nitrite or nitric oxide reductase. 

V. DISSIMILATORY NITRITE REDUCTASES, ENZYMES THAT GENERATE NITRIC OXIDE IN DENITRIFYING BACTERIA... 

Denitrifying bacteria produce one or the other of two usually soluble but very dissimilar nitrite reductases, heme- and Cu-containing types (Brittain et al., 1992 Coyne et al., 1989), both of which are found in the periplasmic space of gram-negative bacteria (Coyne et al., 1990). 

Although the Cu-type family of nitrite reductase is comprised of soluble enzyme and localized in the periplasmic space in gram-negative bacteria, it has proved to be a membrane-bound enzyme in denitrifying Bacillus, which is gram positive and lacks an outer membrane and periplasmic space (Denariaz et ai, 1991 Urata and Satoh, 1991 Ho etal., 1993). 

It was long believed that bacteria were unique in their ability to denitrify. However, Shoun and Tanimoto (1991) and Shoun et al., (1989) demonstrated that the fungus, Fusarium oxysporum, could be induced to synthesize an enzyme system capable of the anaerobic reduction of nitrite to N2O. Induction occurred under conditions of low oxygen concentrations in the presence of nitrate or nitrite. One and pethaps the only component of this nitrite reductase system is a unique, soluble cytochrome P-450 (P-450dNIR), which is more similar in its cDNA-inferred amino acid sequence to soluble, bacterial P-450 enzymes (espe-... 

Calmels, S., Ohshima, H., and Bartsch, H. (1988). Nitrosamine formation by denitrifying and non-denitryfying bacteria Implications of nitrite reductase and nitrate reductase in nitrosation catalysis. . Gen. Microbiol. 134, 221-226. 

Coyne, M. S., Arunakumari, A., Averill, B. A., and Tiedje, J. M. (1989). Immunological identification and distribution of dissimilatory heme cd, and nonheme copper nitrite reductases in denitrifying bacteria. Appl. Environ. Microbiol. 55, 2924-2931. 

Denariaz, G., Payne, W. J., and LeGall, J. (1991). The denitrifying nitrite reductase of Bacillus halodenitrificans. Biochim. Biophys. Acta 1056, 225-232. 

Kakutani, T., Watanabe, H., Arima, K., and Beppu, T. (1981). Purification and properties of a copper-containing nitrite reductase from a denitrifying bacterium, Alcaligenes faecalis strain S-6. J. Biochem. (Tokyo) 89, 453-461. 

Masuko, M., Iwasaki, H., Sakurai, T., Suzuki, S., and Nakahara, A. (1984). Characterization of nitrite reductase from a denitrifier, Alcaligenes. sp. NCIB 11015. A novel copper protein. J. Biochem. (Tokyo) 96, 447-454. 

Moir, W. B., Baratta, D., Richardson, D. J., and Ferguson, S. ]. (1993). The purification of a cd -type nitrite reductase from, and the absence of a copper-type nitrite reductase from, the aerobic denitrifier Thiosphaera pantotropha. Eur. J. Biochem. 212, 377-385. 

Sawada, E., Satoh, T., and Kitamura, H. (1978). Purification and properties of a dissi-milatory nitrite reductase of a denitrifying phototrophic bacterium. Plant Cell Physiol. 19, 1339-1351. 

Shapleigh, J. P., and Payne, W. J. (1985a). Differentiation of cd, cytochrome and copper nitrite reductase production in denitrifiers. FEMS Microbiol. Lett. 26, 275-279. 

Heme d is a chlorin,85 as is acrylochlorin heme from certain bacterial nitrite reductases (Fig. 16-6).86 87 Siroheme (Fig. 16-6), which is found in both nitrite and sulfite reductases of bacteria (Chapter 24) 38/89 is an isobacteriochlorin in which both the A and B rings are reduced. It apparently occurs as an amide siroamide (Fig. 16-6) in Desulfovibrio.90 Heme of nitrite reductases of denitrifying bacteria is a dioxo-bacteriochlorin derivative (Fig. 16-6).91 92... 

A denitrifying nitrite reductase from Achromobacter cycloclastes1533 and Ps. denitrificans1534 is a copper protein, containing two moles copper per mole of protein (molecular weight 69 000). This reduces nitrite to nitrogen monoxide. 

This chapter focuses on the chemistry ofbiomimetic copper nitrosyl complexes relevant to the NO-copper interactions in proteins that are central players in dissimilatory nitrogen oxide reduction (denitrification). The current state of knowledge of NO-copper interactions in nitrite reductase, a key denitrifying enzyme, is briefly surveyed the syntheses, structures, and reactivity of copper nitrosyl model complexes prepared to date are presented and the insight these model studies provide into the mechanisms of denitrification and the structures of other copper protein nitrosyl intermediates are discussed. Emphasis is placed on analysis of the geometric features, electronic structures, and biomimetic reactivity with NO or NOf of the only structurally characterized copper nitrosyls, a dicopper(II) complex bridged by NO and a mononuclear tris(pyrazolyl)hydroborate complex having a Cu(I)-NO formulation. 

Surprisingly, its biological redox partners remain largely unknown. It has been implicated in anaerobic nitrite respiration and it has been shown that azurin can donate electrons to nitrite reductase, a function that is proposed to be carried out by another cupredoxin, pseudoazurin (see Section IV, E). On the other hand, azurin is not an inducible protein and denitrifying bacteria express azurin constitutively under aerobic conditions. 

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