Denitrification pathways

Nitric oxide and NjO are direct intermediates in the denitrification pathway, the reduction of NO3 to Nj. Reduction to Nj is often incomplete, so that both NjO and Nj are equally important end products of denitrification, the ratio of NjO/Nj production being determined by soil physical properties. For example, NjO is the main end-product in acid soils, whereas low redox potentials and high organic matter content favour the further reduction to Nitric... 

Sigman et al. [134] have described a bacterial method for measuring the isotopic composition of seawater nitrate at the natural-abundance level. The method is based on the analysis of nitrous oxide gas (N2O) produced quantitatively from nitrate by denitrifying bacteria. The classical denitrification pathway consists of the stepwise reduction of nitrate (NOp to nitrite (N02), nitric oxide (NO), nitrous oxide (N2O), and dinitrogen (N2) ... 

Each of these steps is carried out by a dedicated enzyme encoded by a distinct gene. There is a rich literature on natural and genetically modified bacterial strains that lack discrete components of the denitrification pathway [ 140]. The... 

Nitrous oxide (N2O) reductases carry out the terminal reduction step in the denitrification pathway. One has been isolated from Pseudomonas... 

As will be discussed further in this chapter, there is now much evidence to suggest that NO is an obligatory intermediate in the denitrification pathway. Furthermore, there is evidence that NH3 nitrifiers can synthesize the denitrification apparatus in addition to the nitrification apparatus and that the former system can produce NO and N2O (also N2 in at least one case) from nitrite under low partial pressures of O2. It is possible therefore that NO may be an intermediate in the denitrification activity of nitrifiers and so arise as a secondary consequence of NH3 oxidation. NO can also be ptoduced by nondenitrifying organisms under certain conditions. For example, NO can be slowly produced by the anaerobic reduction of nitrite, but only in absence of nitrate, by a variety of enteric bacteria. Some of the NO can be further reduced to N2O. 

III. EVIDENCE FOR AND AGAINST NITRIC OXIDE AS AN INTERMEDIATE IN DENITRIFICATION PATHWAY... 

If NO were an obligatory intermediate in the denitrification pathway, then there should exist a separate and specific enzyme to reduce NO to N2O. This enzyme would need to keep pace with the flux of denitrification as set by nitrite... 

IV. NITRIC OXIDE REDUCTASE, NITRIC OXIDE-CONSUMING ENZYME OF DENITRIFICATION PATHWAY... 

Braun, C., and Zumft, W. G. (1991). Marker exchange of the structural genes for nitric oxide reductase blocks the denitrification pathway of Pseudomonas stutzeri at nitric oxide. . Biol. Chem. 266, 22785-22788. 

Figure 2 The dissimilatory denitrification pathway. NO3 is reduced to NO2 by a membrane-bound or periplasmic nitrate reductase(NaR). N02 is reduced to NO by either a cytochrome cdi or copper nitrite reductase (NiR). NO is reduced to N2O by nitric oxide reductase (NOR). N2O is reduced to N2 by nitrous oxide reductase (N2OR). Electron transport from uhiquinol (UQH2) at NaR and the cyt hcj complex is coupled to generation of a proton gradient...

Nitric oxide reductase (Nor) forms part of the denitrification pathway found in Bacteria and Archaea. Denitrification is the five-step conversion of nitrate to dinitrogen according to NOs N02 NO N2O N2. Nor catalyzes the reaction,... 

Figure 6.1 Denitrification pathway involved in canonical denitrification. The four enzymes involved in the sequential reduction of nitrate are show.

Apphcation to the smdy of proteolysis and proteolytic products. Biol. Chem. 9, 185—204. Vance-Harris, C., and Ingah, E. (2005). Denitrification pathways and rates in the sandy sediments of the Georgia continental shelf, USA. Geochem. Trans. 6(1), 12—18. 

It is also possible to use antibody based approaches to assay the accumulation of enzymes involved in denitrification pathways in environmental samples. Ward and Cockroft (1993) developed antisera to nitrite reductase from a marine Pseudomonas stutzeri isolate, and assayed sediment, water column and microbial mat samples. The antibody identified nitrite reductase in mat samples and was more specific than a DNA probe designed against the same species (Ward and Cockroft, 1993). JVIore recently, other strains of denitrifiers have been isolated from both sediment and water column samples. Sequence information regarding the denitrifying genes should prove invaluable for future refinement of both sequence based approaches and the development of additional antibodies to denitrifiers important in the marine environment. 

The anaerobic and aerobic nitrifier denitrification pathways differ in that NO is an end product under anaerobic conditions rather than an intermediate compound. In addition, nitrogen dioxide-dependent NH3 oxidation by N. eutropha does not require ammonium monooxygenase (Schmidt et al., 2002), demonstrating that the two pathways are enzymatically different. In the absence of NH3, N. eutropha can use H2 or simple organic compounds as electron donors (Abeliovich and Vonhak, 1992 Bock et al., 1995). In contrast to the anammox process, which is strictly anaerobic, O2 does not inhibit N02-dependent NH3 oxidation and N2 production can occur even under aerobic conditions (Zart and Bock, 1998). However, Shrestha et al. (2002) observed N2 production... 

N20 can form in natural nitrogen cycles (mainly in soils) through denitrification pathways (Figure l).2,90 The latter involves the NoR enzymes.11 Large emissions appear as a by-product... 

Provided the precursor of the NO-reductases and the cytochrome oxidases was a NO-reductase, it is possible that other enzymes of the denitrification pathway existed. Were this the case, however, it would be possible that the cytochrome oxidases are the product of a combination of various enzymes of this metabolic pathway. The basic structure of the cytochrome oxidases would then have originated from an earlier NO-reductase, in which the CuB-structure of unknown origin had been added. The CuA-dimer could have been added to the cytochrome oxidases later by translocation of a gene. The origin of this gene would probably be that of N20-reductase. If these assumption are correct, aerobic respiration evolved in organisms which were already capable of denitrification. 

Each step of the denitrification pathway is catalyzed by a distinct enzyme, nitrogen oxide reductase (nitrate reductase, nitrite reductase, nitric oxide reductase, and nitrous oxide reductase), that transfers electrons from the chain to the particular intermediate. Thermodynamically, in the absence of oxygen, nitrogen oxides are the most preferred electron acceptors by facultative bacterial groups. The role of nitrogen oxides in regulating organic matter decomposition has been discussed in earlier chapters (see Chapter 5). 

In wetlands, N2O production via denitrification and subsequent emission is governed by the oxygen status of the sediment, supply of nitrifiable nitrogen, moisture content, and temperature. Since the denitrification pathway requires anaerobic conditions, a low partial oxygen pressure during the denitrification process enhances the formation of NjO. 

Alvarez, L., Bricio, C, Gomez, M.J., and Berenguer, J. (2011) Lateral transfer of the denitrification pathway genes among Ihermus thermophilus strains. Appl. Environ. Microbiol., T7, 1352-1358. 

Many bacterial genera contain denitrifying species Achromobacter, Alcaligenes (Alcaligenes odorans denitrifies nitrite). Bacillus, Chromobacterium, Coryne-bacterium, Halobacterium, Hyphomicrobium, Morax-ella, Paracoccus, Pseudomonas, Spirillum, Thiobacil-lus and Xanthomonas. In some species of Pseudomonas and Corynebacterium, N O is the final denitrification product. All these bacteria are aerobes that are able to respire (denitrify) nitrate under anaerobic conditions. The only true anaerobe able to carry out denitrification is Propionibacterium. There is no evidence for other intermediates in the above denitrification pathway, but in the formation of nitrous oxide (NjO) an NN bond must be formed, and there may exist transient enzyme-bound intermediates that have not yet been identified. The enzymology of denitrification from nitrite is poorly understood. It seems likely that each stage is linked to electron transport via a cytochrome system, but sites of ATP synthesis have not been unequivocally located. 

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