Nitrogen fixation is done by bacteria. The bacteria that carry out nitrogen fixation occur in either a free‐living form or as symbionts living in the roots of leguminous plants, such as soybeans, clover, and peas. Nitrogen fixation involves the use of ATP and reducing equivalents derived from primary metabolism. The overall reaction is catalyzed by nitrogenase.
Nitrogenase is a two‐protein complex. One component, called Nitrogenase Reductase (NR) is an iron‐containing protein that accepts electrons from ferredoxin, a strong reductant, and then delivers them to the other component, called Nitrogenase, or Iron‐Molybdenum protein. See Figure 1 .
Nitrogenase first accepts electrons from NR and protons from solution. Nitrogenase binds a molecule of molecular nitrogen (releasing H 2 at the same time), and then accepts electrons and protons from NR, adding them to the N 2 molecule, eventually releasing two molecules of ammonia, NH 3. Release of molecular hydrogen, H 2, apparently is an intrinsic part of nitrogen fixation. Many nitrogen‐fixing systems contain an enzyme, hydrogenase, which harvests the electrons from molecular hydrogen and transfers them back to ferredoxin, thus saving some of the metabolic energy that is lost during nitrogen reduction.
A major part of the energy of photosynthesis in nodulated plants is used for N 2 fixation. At least sixteen ATPs are hydrolyzed during the reduction of a single nitrogen molecule. This drain of the energy from photosynthesis severely limits the growth of plants that fix nitrogen. For example, the yield of useful energy (protein, carbohydrate, and oil) from a field of maize is much greater than from a field of soybeans.
Nitrogenase is extremely sensitive to oxygen. Root nodules of nitrogen‐fixing plants contain an oxygen‐binding protein, leghemoglobin, which protects nitrogenase by binding molecular oxygen.
Similar mechanisms operate in the action of nitrate reductase and nitrite reductase. Both of these substances are produced from ammonia by oxidation. Plants and soil bacteria can reduce these compounds to provide ammonia for metabolism. The common agricultural fertilizer ammonium nitrate, NH 4NO 3, provides reduced nitrogen for plant growth directly, and by providing a substrate for nitrate reduction. NADH or NADPH is the electron donor for nitrate reductase, depending on the organism.
The first step is the reduction of nitrate to nitrate.
Step two involves nitrite reductase reducing nitrite to ammonia.
NO ‐ (nitrite) and NH 2OH (hydroxylamine) are intermediates in the reaction but do not dissociate from nitrite reductase.