Nitrate (anaerobic) Pathway Map

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This pathway was contributed by Jeffrey P. Osborne and Joseph Planer, Manchester College.

As the highest oxidation state of nitrogen and one of the reactive, or "fixed", forms of nitrogen, nitrate can serve both as a source of nitrogen for growth and an electron sink. Under anaerobic conditions, nitrate metabolism occurs in several, competing, microbial processes. Dissimilatory nitrate reduction to ammonia (DNRA) and assimilatory nitrate reduction to ammonia retain the nitrogen in a fixed form, ammonia, keeping it available for further biological processes (Zumft, 1997). In contrast, denitrification (Zumft, 1997) ultimately transforms nitrate to dinitrogen (N2), a gas that removes the nitrogen from the habitat, unless/until nitrogen fixation once again fixes the nitrogen. In the presence of nitrite, the anaerobic ammonium oxidation (anammox) process also produces N2, by effectively combining nitrite with ammonia (Keunen, 2008). DNRA, denitrification, and anammox processes result in energy conservation and provide an electron sink. DNRA also functions to remove excess fixed nitrogen from an organism. Assimilatory nitrate reduction provides ammonia for biosynthesis of nitrogen-containing compounds (Zumft, 1997; Kraft et al., 2011).

Nitrate reduction by microorganisms is a major biogeochemical process. The flux through denitrification in the ocean is estimated to be 450 Tg N y-1 (Codispoti, LA, Brandes JA, Christensen, JP, Devol, AH, Naqvi, SWA, Paerl, HW and Yoshinari T, Scientia Marina, 65:Suppl.2, 85-105). In addition, the anammox pathway accounts for roughly 50% of fixed nitrogen turnover in marine environments (Arrigo et al., 2005). Nitrate concentrations are also an important issue in agricultural fertilizer use and wastewater treatment. Although plants can use nitrate as a nitrogen source, excess nitrate in bodies of water and in drinking water supplies can cause algal blooms and lead to public health problems, respectively (Kraft et al., 2011).

The first step of the nitrate degradation pathway is the two electron reduction of nitrate to nitrite, which is accomplished by three classes of nitrate reductases in bacteria and archaea. Assimilatory nitrate reductases (NAS) are usually cytoplasmic and enable microbes to use environmental nitrate as a nitrogen source. Periplasmic nitrate reductases (NAP) perform redox balancing, scavenge nitrate in nitrate-limited environments, and serve in aerobic or anaerobic denitrification. Found in the membrane, respiratory nitrate reductases (NAR) enable the use of nitrate as a terminal electron sink in DNRA and in anaerobic denitrification (González et al., 2006).

Subsequently, nitrite can be reduced directly to ammonia, the most negative oxidation state of nitrogen. This reaction is catalyzed by cytochrome c nitrite reductase (NrfA) or octoheme cytochrome c nitrite reductase in DNRA and by NAD(P)H-dependent nitrite reductase (NirB) in nitrite assimilation (Kraft et al., 2011).

In denitrification, nitrite is reduced to nitric oxide in a reaction catalyzed by copper nitrite reductase (NirK). The resulting nitric oxide is then reduced to nitrous oxide by nitric oxide reductase. Next, nitrous oxide is reduced to N2 by nitrous oxide reductase. Note that the release to the environment of nitrous oxide, a potent greenhouse gas and ozone layer depleter, can occur before the final reduction step to N2 (Zumft, 1997).

In anammox, nitrite is also reduced to nitric oxide, but the reaction is catalyzed by cytochrome cd1 nitrite reductase (NirS). Then, the relatively oxidized nitric oxide is combined with ammonia, which is comparatively reduced, to produce hydrazine in a reaction catalyzed by hydrazine synthase. Hydrazine dehydrogenase next removes electrons and the protons from hydrazine to make N2 (Kartal et al., 2011). Found in the bacterial group Planctomycetes, annamox chemistry occurs in a specialized organelle, the anammoxosome, which has a ladderane lipid membrane, probably structured to protect the cell from the highly reactive pathway intermediates (Francis et al., 2007).

Finally, N2 from denitrification or anammox can be reduced to ammonia by nitrogenase in the energy intensive process of nitrogen fixation, catalyzed by nitrogenase (Zumft, 1975).

The following is a text-format nitrate (anaerobic) pathway map. One of the organisms that can initiate the pathway is given, but other organisms can also carry out later steps. Follow the links for more information on compounds or reactions. This map is also available in graphic (12k) format.

      Paracoccus denitrificans
nitrate reductase | nitrate reductase 
           (NADH) | (cytochrome) 
      ferredoxin- | nitrate reductase 
nitrate reductase | 
                  |           cytochrome cd1
                  V          nitrite reductase
          Escherichia coli                           |
                  |                                  V
                  |                             Nitric oxide
                  |                      Parococcus     Kuenenia
                  |                    denitrificans  stuttgartiensis
                  |                            /            \
                  |                           /              \
                  |             nitric oxide /                \ hydrazine
          NAD(P)H |               reductase /                  \ synthase
nitrite reductase |                        /                    \
                  |                       V                      V
     cytochrome c |                 Nitrous oxide            Hydrazine
nitrite reductase |                        \                     /
                  |                         \                   /
                  |                          \                 /
                  |             nitrous oxide \               / hydrazine
                  |                  reductase \             / dehydrogenase
                  |                             \           /
                  |                              \         /
                  |                               V       V
                  |                               Nitrogen 
                  |                       Bradyrhizobium japonicum
                  |                                  |
                  V           nitrogenase            |
              Ammonia <------------------------------+

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Page Author(s): Jeffrey P. Osborne, Manchester College

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