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Nitric Oxide Reductase

Figure 1. The Nitrogen Cycle. Nitric oxide (NO) and nitrous oxide (N2O) are intermediates in the denitrification of nitrate (NO3-) to nitrogen gas (N2). Nitric oxide reductase reduces NO to N2O.

EC 1.7.2.5

Biological Importance of Nitric Oxide Reductase (NOR)

Through the process of denitrification, see Figure 1, organisms reduce nitrate (NO3-) to nitrogen gas (N2). Two important intermediates of the reduction pathway are nitric oxide (NO) and nitrous oxide (N2O). The reducing reaction that transforms NO into N2O is catalyzed by nitric oxide reductase (NOR).

Type of NO Reductases

Identified bacterial NORs include cNOR, qNOR, and qCuNOR. cNOR was found in denitrifying bacteria: Paracoccus denitrificans, Halomonas halodenitrificans, Pseudomonas nautica, Pseudomonas stutzeri, and Pseudomonas aeruginosa. cNOR was first isolated from P. aeruginosa. qNOR was isolated from Geobacillus stearothermophilus.

Structure

Components

NOR is made up of two subunits, NorC (small) and NorB (large), with a binuclear iron centre. The binuclear iron center is the active site. It is comprised of two b-type hemes and a non-heme iron (FeB). The ligands are connected through a μ-oxo bridge. Histidine (His) residues are attached to the heme b3 in the small subunit. The hydrophilic region of the larger subunit has His and methionine (Met) ligands. Structure is similar to cytochrome oxidases.

The active site is conserved between cNOR and qNOR, although differences (ie. heme type) occur between cNOR and qNOR.

Folding

Enzymatic folding produced 13 alpha-helices (12 from NorB, 1 from NorC) located within and through the membrane. The folded metalloenzyme transverses the membrane.

Pathway

2NO + 2e- + 2H = 2N2O + H20

Inputs: 2 molecules of NO, 2 electrons, 2 protons

Outputs: 1 molecule of N2O an1 molecule of H2O

Mechanism

The mechanism of catalysis is still unknown, although hypotheses exist.

Cordas et al. 2013 proposes three options: the trans-mechanism, the cis-FeB and the cis-heme b3 mechanisms.

Based on the structure of the enzyme, Shiro 2012 proposes the following mechanism: (1) NO molecules bind at the binuclear center, (2) electrons are transferred from the ferrous irons to the NO, (3) charged NO molecules have the potential to form N to N bonds, and (4) N to O bonds are potentially broken by water, allowing for the N2O and H2O to be released.

According to Hino et al. 2010, the changing charge of the active site causes NO to bind, form N2O and leave the enzyme. Because the active site is positioned near two hydrogen bound Glu, which provide an electron-negative charge. Electro-negative charge reduces the reaction potential for heme b3. NO is able to bind to the binuclear activation site. Glutamic acid (Glu) residues provide protons needed for removal of N2O and production of H2O.

Function

NOR allows for the formation of a nitrogen to nitrogen (N--N)  bond. The conformation changes of the active site and attached ligands (ie. Glu211) allows NO to be positioned in the crowded binuclear center in position for N--N bonds to form.

NO is quickly reduced to N2O to prevent cellular toxicity. N2O, a potent greenhouse gas, is released.

Organisms

Bacteria, archaea and fungi use Nitric Oxide Reductase. qNOR is found in denitrifying bacteria and archaea, as well as pathogenic bacteria not involved in denitrification. Denitrifying fungi reduce NO using P-450nor soluble enzyme.

Bibliography

Collman, J. P., Yang, Y., Dey, A., Decreau, R. A., Ghosh, S., Ohta, T., & Solomon, E. I. (2008). A functional nitric oxide reductase model. Proceedings of the National Academy of Sciences, 105(41), 15660–15665. https://doi.org/10.1073/pnas.0808606105

Cordas, C. M., Duarte, A. G., Moura, J. J. G., & Moura, I. (2013). Electrochemical behaviour of bacterial nitric oxide reductase—Evidence of low redox potential non-heme FeB gives new perspectives on the catalytic mechanism. Biochimica et Biophysica Acta (BBA) - Bioenergetics, 1827(3), 233–238. https://doi.org/10.1016/j.bbabio.2012.10.018

Hendriks, J., Oubrie, A., Castresana, J., Urbani, A., Gemeinhardt, S., & Saraste, M. (2000). Nitric oxide reductases in bacteria. Biochimica et Biophysica Acta (BBA) - Bioenergetics, 1459(2–3), 266–273. https://doi.org/10.1016/S0005-2728(00)00161-4

Hino, T., Matsumoto, Y., Nagano, S., Sugimoto, H., Fukumori, Y., Murata, T., … Shiro, Y. (2010). Structural Basis of Biological N2O Generation by Bacterial Nitric Oxide Reductase. Science, 330(6011), 1666–1670. https://doi.org/10.1126/science.1195591

Shiro, Y. (2012). Structure and function of bacterial nitric oxide reductases. Biochimica et Biophysica Acta (BBA) - Bioenergetics, 1817(10), 1907–1913. https://doi.org/10.1016/j.bbabio.2012.03.001

Yeung, N., Lin, Y.-W., Gao, Y.-G., Zhao, X., Russell, B. S., Lei, L., … Lu, Y. (2009). Rational design of a structural and functional nitric oxide reductase. Nature, 462(7276), 1079–1082. https://doi.org/10.1038/nature08620