The nitrogen in amino acids, purines, pyrimidines, and other biomolecules ultimately comes from atmospheric nitrogen, N₂. The biosynthetic process starts with the reduction of N₂ to NH₃ (ammonia), a process called nitrogen fixation. The extremely strong N ≡ N bond, which has a bond energy of 940 kJ mol⁻¹ (225 kcal mol⁻¹), is highly resistant to chemical attack. Indeed, Antoine Lavoisier named nitrogen gas “azote,” from Greek words meaning “without life,” because it is so unreactive. Nevertheless, the conversion of nitrogen and hydrogen to form ammonia is thermodynamically favorable; the reaction is difficult kinetically because of the activation energy required to form intermediates along the reaction pathway.

Although higher organisms are unable to fix nitrogen, this conversion is carried out by some bacteria and archaea. Symbiotic Rhizobium bacteria invade the roots of leguminous plants and form root nodules in which they fix nitrogen, supplying both the bacteria and the plants. The importance of nitrogen fixation by diazotrophic (nitrogen-fixing) microorganisms to the metabolism of all higher eukaryotes cannot be overstated: the amount of N₂ fixed by these species has been estimated to be 10¹¹ kilograms per year, about 60% of Earth’s newly fixed nitrogen. Lightning and ultraviolet radiation fix another 15%; the other 25% is fixed by industrial processes. The industrial process for nitrogen fixation devised by Fritz Haber in 1910 is still being used in fertilizer factories.

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The fixation of N₂ is typically carried out by mixing with H₂ gas over an iron catalyst at about 500°C and a pressure of 300 atmospheres.

To meet the kinetic challenge, the biological process of nitrogen fixation requires a complex enzyme with multiple redox centers. The nitrogenase complex, which carries out this fundamental transformation, consists of two proteins: a reductase (also called the iron protein or Fe protein), which provides electrons with high reducing power, and nitrogenase (also called the molybdenum–iron protein or MoFe protein), which uses these electrons to reduce N₂ to NH₃. The transfer of electrons from the reductase to the nitrogenase component is coupled to the hydrolysis of ATP by the reductase (Figure 24.1).

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 mol 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, generated by oxidative processes. 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.

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 (Section 6.3).

Both the reductase and the nitrogenase components of the complex are iron–sulfur proteins, in which iron is bonded to the sulfur atom of a cysteine residue and to inorganic sulfide. Recall that iron–sulfur clusters act as electron carriers (Section 18.3). The reductase is a dimer of identical 30-kDa subunits bridged by a 4Fe–4S cluster (Figure 24.2).

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The role of the reductase is to transfer electrons from a suitable donor, such as reduced ferredoxin, to the nitrogenase component. The 4Fe–4S cluster carries the electrons, one at a time, to nitrogenase. The binding and hydrolysis of ATP triggers a conformational change that moves the reductase closer to the nitrogenase component, whence it is able to transfer its electron to the center of nitrogen reduction. The structure of the ATP-binding region reveals it to be a member of the P-loop NTPase family (Section 9.4) that is clearly related to the nucleotide-binding regions found in G proteins and related proteins. Thus, we see another example of how this domain has been recruited in evolution because of its ability to couple nucleoside triphosphate hydrolysis to conformational changes.

The nitrogenase component is an α₂β₂ tetramer (240 kDa), in which the α and β subunits are homologous to each other and structurally quite similar (Figure 24.3). The nitrogenase 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), donated by S-adenosylmethionine, 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.

Electrons from the reductase enter at the P clusters, which are located at the α–β interface. The role of the P clusters is to store electrons until they can be used productively to reduce nitrogen at the FeMo cofactor. The FeMo cofactor is the site of nitrogen fixation. One face of the FeMo cofactor is likely to be the site of nitrogen reduction. The electron-transfer reactions from the P cluster take place in concert with the binding of hydrogen ions to nitrogen as it is reduced. Further studies are under way to elucidate the mechanism of this remarkable reaction.

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The next step in the assimilation of nitrogen into biomolecules is the entry of NH₄⁺ into amino acids. The amino acids glutamate and glutamine play pivotal roles in this regard, acting as nitrogen donors for most amino acids. The α-amino group of most amino acids comes from the α-amino group of glutamate by transamination (Section 23.3). Glutamine, the other major nitrogen donor, contributes its side-chain nitrogen atom in the biosynthesis of a wide range of important compounds, including the amino acids tryptophan and histidine.

Glutamate is synthesized from NH₄⁺ and α-ketoglutarate, a citric acid cycle intermediate, by the action of glutamate dehydrogenase. We have already encountered this enzyme in the degradation of amino acids (Section 23.3). Recall that NAD⁺ is the oxidant in catabolism, whereas NADPH is the reductant in biosyntheses. Glutamate dehydrogenase is unusual in that it does not discriminate between NADH and NADPH, at least in some species.

The reaction proceeds in two steps. First, a Schiff base forms between ammonia and α-ketoglutarate. The formation of a Schiff base between an amine and a carbonyl compound is a key reaction that takes place at many stages of amino acid biosynthesis and degradation.

Schiff bases are easily protonated. In the second step, the protonated Schiff base is reduced by the transfer of a hydride ion from NAD(P)H to form glutamate.

This reaction is crucial because it establishes the stereochemistry of the α-carbon atom (S absolute configuration) in glutamate. The enzyme binds the α-ketoglutarate substrate in such a way that hydride transferred from NAD(P)H is added to form the l isomer of glutamate (Figure 24.4). As we shall see, this stereochemistry is established for other amino acids by transamination reactions that rely on pyridoxal phosphate.

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 acylphosphate intermediate, which then reacts with ammonia to form glutamine.

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A high-affinity ammonia-binding site is formed in the enzyme only after the formation of the acylphosphate intermediate. A specific site for ammonia binding is required to prevent attack by water from hydrolyzing the intermediate and wasting a molecule of ATP. The regulation of glutamine synthetase plays a critical role in controlling nitrogen metabolism (Section 24.3).

Glutamate dehydrogenase and glutamine synthetase are present in all organisms. Most prokaryotes also contain an evolutionarily unrelated enzyme, glutamate synthase, which catalyzes the reductive amination of α-ketoglutarate to glutamate. Glutamine is the nitrogen donor.

The side-chain amide of glutamine is hydrolyzed to generate ammonia within the enzyme, a recurring theme throughout nitrogen metabolism. When NH₄⁺ is limiting, most of the glutamate is made by the sequential action of glutamine synthetase and glutamate synthase. The sum of these reactions is

Note that this stoichiometry differs from that of the glutamate dehydrogenase reaction in that ATP is hydrolyzed. Why do prokaryotes sometimes use this more expensive pathway? The answer is that the value of K_M of glutamate dehydrogenase for NH₄⁺ is high (∼ 1 mM), and so this enzyme is not saturated when NH₄⁺ is limiting. In contrast, glutamine synthetase has very low K_M for NH₄⁺. Thus, ATP hydrolysis is required to capture ammonia when it is scarce.

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