The nitrogen needed for amino acids (and for the heterocyclic bases of nucleotides) comes from two major sources: nitrogen gas in the atmosphere and nitrate (NO3⊖) in soil and water. Atmospheric N2 which constitutes about 80% of the atmosphere, is the ultimate source of biological nitrogen. This molecule can be metabolized, or fixed, by only a few species of bacteria. N2 and NO3⊖ must be reduced to ammonia in order to be used in metabolism. The ammonia produced is incorporated into amino acids via glutamate, glutamine, and carbamoyl phosphate.
N2 is chemically unreactive because of the great strength of the triple bond (N≡N). Some bacteria have a very specific, sophisticated enzyme—nitrogenase1—that can catalyze the reduction of N2 to ammonia in a process called nitrogen fixation. In addition to biological nitrogen fixation there are two additional nitrogen-converting processes. During lightning storms, high-voltage discharges cause the oxidation of N2 to nitrate and nitrite (NO2⊖). Industrially, nitrogen is converted to ammonia for use in plant fertilizers by an energetically expensive process that requires high temperature and pressure as well as special catalysts to drive the reduction of N2 by H2. The availability of biologically useful nitrogen is often a limiting factor for plant growth, and the application of nitrogenous fertilizers is important for obtaining high crop yields. Although only a small percentage of the nitrogen undergoing metabolism comes directly from nitrogen fixation, this process is the only way that organisms can use the huge pool of atmospheric N2.
The overall scheme for the interconversion of the major nitrogen-containing compounds is shown in Figure 17.1. The flow of nitrogen from N2 to nitrogen oxides, ammonia, and nitrogenous biomolecules and then back to N2 is called the nitrogen cycle. Most of the nitrogen shuttles between ammonia and nitrate. Ammonia from decayed organisms is oxidized by soil bacteria to nitrate. This formation of nitrate is called nitrification. Some anaerobic bacteria can reduce nitrate or nitrite to N2 (denitrification). Most green plants and some microorganisms contain nitrate reductase and nitrite reductase, enzymes that together catalyze the reduction of nitrogen oxides to ammonia.
This ammonia is used by plants, which supply amino acids to animals. Reduced ferredoxin (formed in the light reactions of photosynthesis) is the source of the reducing power in plants and photosynthetic bacteria.
Let’s examine the enzymatic reduction of N2. Most nitrogen fixation in the biosphere is carried out by bacteria that synthesize the enzyme nitrogenase. This multisubunit protein catalyzes the conversion of each molecule of N2 to two molecules of NH3 (ammonia). Nitrogenase is present in various species of Rhizobium and Bradyrhizobium that live symbiotically in root nodules of many leguminous plants, including soybeans, peas, alfalfa, and clover (Figure 17.2). N2 is also fixed by freeliving soil bacteria such as Agrobacteria, Azotobacter, Klebsiella, and Clostridium and by cyanobacteria (mostly Trichodesmium spp.) found in the ocean. Most plants require a supply of fixed nitrogen from sources such as decayed animal and plant tissue, nitrogen compounds excreted by bacteria, and fertilizers. Vertebrates obtain fixed nitrogen by ingesting plant and animal matter.
Nitrogenase is a protein complex that consists of two different polypeptide subunits with a relatively complicated electron-transport system. One polypeptide (called iron protein) contains a [4 Fe–4 S] cluster, and the other (called iron–molybdenum protein) has two oxidation–reduction centers, one containing iron in an [8 Fe–7 S] cluster, and the other containing both iron and molybdenum. Nitrogenases must be protected from oxygen because the metalloproteins are highly susceptible to inactivation by O2. For example, strict anaerobes carry out nitrogen fixation only in the absence of O2. Within the root nodules of leguminous plants, the protein leghemoglobin (a homolog of vertebrate myoglobin) binds and thereby keeps its concentration sufficiently low in the immediate environment of the nitrogen-fixing enzymes of rhizobia. Nitrogen fixation in cyanobacteria is carried out in specialized cells (heterocysts) whose thick membranes inhibit entry of O2.
A strong reducing agent—either reduced ferredoxin or reduced flavodoxin (a flavoprotein electron carrier in microorganisms)—is required for the enzymatic reduction of N2 to NH3. An obligatory reduction of 2 H⊕ to H2 accompanies the reduction of N2. For each electron transferred by nitrogenase, at least two ATP molecules must be converted to ADP and Pi (inorganic phosphate) so the six-electron reduction of a single molecule of N2 (plus the two-electron reduction of 2 H⊕) consumes a minimum of 16 ATP.
In order to obtain the reducing power and ATP required for this process, symbiotic nitrogen-fixing microorganisms rely on nutrients obtained through photosynthesis carried out by the plants with which they are associated.
©Laurence A. Moran and Pearson/Prentice Hall
1. Monday's Molecule #66
[Nitrogenase Image Credit: Dixon and Kahn (2004) based on the structure PDB 1n2c by Schindelin et al. (1997)]
Dixon, R. and Kahn, D. (2004) Genetic regulation of biological nitrogen fixation. Nature Reviews Microbiology 2, 621-631. doi:10.1038/nrmicro954
Horton, H.R., Moran, L.A., Scrimgeour, K.G., perry, M.D. and Rawn, J.D. (2006) Principles of Biochemisty. Pearson/Prentice Hall, Upper Saddle River N.J. (USA)
Schindelin, H., Kisker, C., Schlessman, J.L., Howard, J.B. and Rees, D.C. (1997) Structure of ADP x AIF4(-)-stabilized nitrogenase complex and its implications for signal transduction. Nature 387: 370-376 [PubMed]