The natural cycle of denitrification comprises a cascade of different enzymes that stepwise reduce nitrate to dinitrogen [1]-[3]:

Hence, denitrification corresponds to the part of the biological nitrogen cycle that is opposed to nitrogen fixation. The reduction of nitric oxide to nitrous oxide is mediated in nature by the NO reductases (NOR), which occur in bacteria (NorBC) as well as in fungi (P450nor). The principal reaction scheme of NO reduction corresponds to the equation:

Bacterial and fungal NORs are fundamentally different in the nature of their active sites. Correspondingly, very different reaction mechanisms have been proposed for these two classes of enzymes (see below).

In the biological denitrification cycle, nitrous oxide is reduced to dinitrogen by the N2O reductases (N2OR) following the principal reaction scheme:

The different classes of NO and N2O reductases are described in more detail in the following sections.

Bacterial NORs (NorBC)
These enzymes are closely related to the respiratory heme-copper oxydases, the so called Cytochrome c-Oxidases (CCO). Therefore, the known crystal structures of different CCOs can serve as a blueprint for the structure of the active center of bacterial NOR. Figure 1 shows the catalytically active site of bovine heart CCO[4], which consists of a high-spin heme a3 and the so called CuB center. The latter one is coordinated by three histidines. The catalytically active center of the NORs has a similar structure and in the reduced state consists of a five-coordinate high-spin heme b (with axial histidine) and a non-heme iron, which substitutes CuB in the active site of the CCOs. Hence, it is designated as FeB.

As in the case of the CCOs, not much is known about the molecular mechanism of the NORs. Analysis of kinetic data of NOR lead to the postulation of the mechanism in Figure 2 [5]. First, two molecules of NO are bound to the two iron centers and then coupled through their nitrogen atoms. After the concomitant release of the product N2O, the two oxidized iron centers are then coupled by an oxo bridge. However, none of these intermediates has been characterized yet. Hence, this reaction mechanism has to be considered as speculation. Alternatively, the reduction of NO could also proceed at FeB only. A corresponding mechanism has been presented for the reaction of NO with CCOs, where two molecules of NO are proposed to bind to the CuB center [6]. Vice versa, it also seems possible that the reaction proceeds solely at the heme b, as has been shown for the model system [Fe(TPP)(NO)] (TPP = tetraphenylporphyrin) [7].

Fungal NORs (P450nor)
In contrast to the bacterial NORs, which are related to the CCOs, the fungal NORs are derived from Cytochrome P450 and hence, are designated as P450nor[8]. The crystal structure of the enzyme from Fusarium oxysporum shows a heme b with the typical axial cysteine thiolate ligand [9]. Several mechanistic studies have shown that the Fe(III) form is catalytically active in the case of P450nor. Coordination of NO then leads to a six-coordinate low-spin complex, that has been characterized with different spectroscopic methods. Figure 3 shows a possible mechanism, which is based on kinetic investigations [10]. In this proposal, the Fe(III)-NO complex is initially reduced by two electrons and the resulting species (intermediate 'I') then reacts with a second molecule of NO forming the product N2O.

The crystal structure of N2O reductase (N2OR) has been determined for two different organisms. N2OR contains a unique copper cluster, CuZ [11], which is the designated site of bonding and reduction of the N2O molecule. As shown in Figure 4, the CuZ cluster consists of four copper centers, which are bridged by a sulfide ion. However, neither the mode of bonding of N2O to CuZ nor the mechanism of reduction to N2 are known [12],[13].

[1] Ferguson, S. J. Curr. Opin. Chem. Biol. 1998, 2, 182-193.

[2] Richardson, D. J.; Watmough, N. J. Curr. Opin. Chem. Biol. 1999, 3, 207-219.

[3] Moura, I.; Moura, J. J. G. Curr. Opin. Chem. Biol. 2001, 5, 168-175.

[4] Tsukihara, T.; Aoyama, H.; Yamashita, E.; Tomizaki, T.; Yamaguchi, H.; Shinzawa-Itoh, K.; Nakashima, R.; Yaono, R.; Yoshikawa, S. Science 1996, 272, 1136-1144.

[5] Zumft, W. J. Inorg. Biochem. 2005, 99, 194-215.

[6] Butler, C. S.; Seward, H. E.; Greenwood, C.; Thomson, A. J. Biochemistry 1997, 36, 16259-16266.

[7] Lin, R.; Farmer, P. J. J. Am. Chem. Soc. 2001, 123, 1143-1150.

[8] Daiber, A.; Shoun, H.; Ullrich, V. J. Inorg. Biochem. 2005, 99, 185-193.

[9] Park, S.-Y.; Shimizu, H.; Adachi, S.-I.; Nakagawa, A.; Tanaka, I.; Nakahara, K.; Shoun, H.; Obayashi, E.; Nakamura, H.; Iizuka, T.; Shiro, Y. Nature Struct. Biol. 1997, 4, 827-832.

[10] Shiro, Y.; Fujii, M.; Iizuka, T.; Adachi, S.-I.; Tsukamoto, K.; Nakahara, K.; Shoun, H. J. Biol. Chem. 1995, 270, 1617-1623.

[11] ) Haltia, T.; Brown, K.; Tegoni, M.; Cambillau, C.; Saraste, M.; Mattila, K.; Djinovic-Carugo, K. Biochem. J. 2003, 369, 77-88.

[12] Paulat, F.; Kuschel, T.; Näther, C.; Praneeth, V. K. K.; Sander, O.; Lehnert, N. Inorg. Chem. 2004, 43, 6979-6994.

[13] Chen, P.; Gorelsky, S. I.; Ghosh, S.; Solomon, E. I. Angew. Chem. Int. Ed. 2004, 43, 4132-4140.