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. 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 amount of N2 fixed by diazotrophic (nitrogen-fixing) microorganisms 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.
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. The extremely strong N![[equivalent]](corehtml/pmc/pmcents/equiv.gif)
N bond, which has a bond energy of 225 kcal mol-1, is highly resistant to chemical attack. Indeed, Lavoisier named nitrogen gas “azote,” 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 intermediates along the reaction pathway are unstable.
Electrons flow from ferredoxin to the reductase (iron protein, or Fe protein) to nitrogenase (molybdenum-iron protein, or MoFe protein) to reduce nitrogen to ammonia. ATP hydrolysis within the reductase drives conformational changes necessary for the efficient transfer of electrons.
). The nitrogenase complex is exquisitely sensitive to inactivation by O2. Leguminous plants maintain a very low concentration of free O2 in their root nodules by binding O2 to leghemoglobin, a homolog of hemoglobin (Section 7.3.1).
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 mole of NN. 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 photosynthesis or 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.
Again, ATP hydrolysis is not required to make nitrogen reduction favorable thermodynamically. Rather, it is essential to reduce the heights of activation barriers along the reaction pathway, thus making the reaction kinetically feasible.
This protein is a dimer composed of two polypeptide chains linked by a 4Fe-4S cluster. Each monomer is a member of the P-loop NTPase family and contains an ATP-binding site.
).
The role of the reductase is to transfer electrons from a suitable donor, such as reduced ferredoxin, to the nitrogenase component. The binding and hydrolysis of ATP triggers a conformational change that moves the reductase closer to the nitrogenase component from 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 9.4.1) that is clearly related to the nucleotide-binding regions found in G proteins and related proteins (Section 15.1.2). 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.
This protein is an heterotetramer composed of two α subunits (red) and two β subunits (blue). The protein contains two copies each of two types of clusters: P clusters and FeMo cofactors. Each P cluster contains eight iron atoms and seven sulfides linked to the protein by six cysteinate residues. Each FeMo cofactor contains one molybdenum atom, seven iron atoms, nine sulfides, and a homocitrate, and is linked to the protein by one cysteinate residue and one histidine residue.
). Electrons enter at the P clusters, which are located at the α-β interface. These clusters are each composed of eight iron atoms and seven sulfide ions. In the reduced form, each cluster takes the form of two 4Fe-3S partial cubes linked by a central sulfide ion. Each cluster is linked to the protein through six cysteinate residues. Electrons flow from the P cluster to the FeMo cofactor, a very unusual redox center. Because molybdenum is present in this cluster, the nitrogenase component is also called the molybdenum-iron protein (MoFe protein). The FeMo cofactor consists of two M-3Fe-3S clusters, in which molybdenum occupies the M site in one cluster and iron occupies it in the other. The two clusters are joined by three sulfide ions. The FeMo cofactor is also coordinated to a homocitrate moiety and to the α subunit through one histidine residue and one cysteinate residue. This cofactor is distinct from the molybdenum-containing cofactor found in sulfite oxidase and apparently all other molybdenum-containing enzymes except nitrogenase.
The FeMo cofactor contains an open center that is the likely site of nitrogen binding and reduction.
). The formation of multiple Fe-N interactions in this complex weakens the NN bond and thereby lowers the activation barrier for reduction.
The next step in the assimilation of nitrogen into biomolecules is the entry of NH4+ into amino acids. Glutamate and glutamine play pivotal roles in this regard. The α-amino group of most amino acids comes from the α-amino group of glutamate by transamination (Section 23.3.1). 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 23.3.1). 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 can be easily protonated. With glutamate dehydrogenase, the protonated Schiff base is reduced by the transfer of a hydride ion from NADPH to form glutamate.
In the active site of glutamate dehydrogenase, hydride transfer from NAD(P)H to a specific face of the achiral protonated Schiff base of α-ketoglutarate establishes the l configuration of glutamate.
). 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 acyl-phosphate intermediate, which then reacts with ammonia to form glutamine.
A high-affinity ammonia-binding site is formed only after the formation of the acyl-phosphate 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 24.3.2).
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 with the use of glutamine as 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 KM 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 high affinity for NH4+. Thus, ATP hydrolysis is required to capture ammonia when it is scarce.
