To meet the kinetic challenge of nitrogen fixation, nitrogen-fixing organisms employ a complex enzyme with multiple oxidation–reduction centers that enable it to reduce the inactive N₂ gas. The nitrogenase complex, which carries out this fundamental transformation, consists of two proteins: a reductase, which provides electrons with high reducing power, and nitrogenase, which uses these electrons to reduce N₂ to NH₃. The transfer of electrons from the reductase to the nitrogenase component requires the hydrolysis of ATP by the reductase (Figure 31.2).

In principle, the reduction of N₂ to NH₃ is a six-electron process:

However, the biological reaction always generates at least 1 mol of H₂ in addition to 2 mol of NH₃ for each mole of N ≡ N. Hence, an input of two additional electrons is required:

In most nitrogen-fixing microorganisms, the eight high-potential electrons come from reduced ferredoxin. Two molecules of ATP are hydrolyzed for each electron transferred. Thus, at least 16 molecules of ATP are hydrolyzed for each molecule of N₂ reduced:

The product of the reduction, NH₃, is a base in aqueous solutions, attracting a proton to form NH₄⁺.

Note that O₂ is required for oxidative phosphorylation to generate the ATP necessary for nitrogen fixation. However, the nitrogenase complex is exquisitely sensitive to inactivation by O₂. To allow ATP synthesis and nitrogenase to function simultaneously, leguminous plants maintain a very low concentration of free O₂ in their root nodules, the location of the nitrogenase. This is accomplished by binding O₂ to leghemoglobin, a homolog of hemoglobin.

Both the reductase and the nitrogenase components of the complex are iron–sulfur proteins, a type of electron carrier that we have seen many times in our study of biochemistry—for instance, in the electron-transport chains of oxidative phosphorylation and photosynthesis. The reductase (also called the iron protein or the Fe protein) is a dimer of identical 30-kDa subunits bridged by a 4Fe-4S cluster (Figure 31.3). The role of the reductase is to transfer electrons from a suitable donor, such as reduced ferredoxin, to the nitrogenase component.

The nitrogenase component, an α₂β₂ tetramer (240 kDa), requires the FeMo cofactor, which consists of [Fe₄-S₃] and [Mo-Fe₃-S₃] subclusters joined by three disulfide bonds. A carbon atom (the interstitial carbon) sits at the interstices of the iron atoms of the FeMo cofactor. The FeMo cofactor is also coordinated to a homocitrate moiety and to the α subunit through one histidine residue and one cysteinate residue (Figure 31.4).

Electrons flow from the reductase to the nitrogenase at a part of the nitrogenase called the P cluster, an iron- and sulfur-rich site. From there, the electrons progress to the FeMo cofactor, the redox center in the nitrogenase. Because molybdenum is present in this cluster, the nitrogenase component is also called the molybdenum–iron protein or MoFe protein. The MoFe cofactor is the site of nitrogen fixation—the conversion of N₂ into ammonia.

The next task in the assimilation of nitrogen into biomolecules is to incorporate ammonium ion (NH₄⁺) into the biochemically versatile amino acids. α-Ketoglutarate and glutamate play pivotal roles as the acceptor of ammonium ion, forming glutamate and glutamine, respectively. Glutamate is synthesized from NH₄⁺ and α-ketoglutarate, a citric acid cycle intermediate, by the action of glutamate dehydrogenase:

Recall that glutamate dehydrogenase also plays a key role in the removal of nitrogen from biological systems.

A second ammonium ion is incorporated into glutamate to form glutamine by the action of glutamine synthetase. This amidation is driven by the hydrolysis of ATP:

ATP participates directly in the reaction by phosphorylating the side chain of glutamate to form an acyl-phosphate intermediate, which then reacts with ammonia to form glutamine.

Glutamate subsequently donates its α-amino group to various ketoacids by transamination reactions to form most of the amino acids. Glutamine, the other major nitrogen donor, contributes its side-chain nitrogen atom in the biosynthesis of a wide range of important compounds.