Nitrogen Fixation Microorganisms Use ATP and a Powerful Reductant to Reduce Atmospheric Nitrogen to Ammonia

Figure 24.1. Aminotransferase Protein Family. A superposition of the structures of four aminotransferases taking part
in amino acid biosynthesis reveals a common fold, indicating that these proteins descended from a common
ancestor.
III. Synthesizing the Molecules of Life
24. The Biosynthesis of Amino Acids
Nitrogen is a key component of amino acids. The atmosphere is rich in nitrogen gas (N2), a very unreactive molecule.
Certain organisms such as bacteria that live in the root nodules of yellow clover (photo at left) can convert nitrogen gas
into ammonia. Ammonia can then be used to synthesize first glutamate and then other amino acids. [(Left) Runk/
Schoenberger from Grant Heilman]
III. Synthesizing the Molecules of Life
24. The Biosynthesis of Amino Acids
24.1. Nitrogen Fixation: Microorganisms Use ATP and a Powerful Reductant to
Reduce Atmospheric Nitrogen to Ammonia
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 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.
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, 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 is coupled to the hydrolysis of ATP
by the reductase (Figure 24.2). 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
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
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 N reduced.
2
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.
24.1.1. The Iron-Molybdenum Cofactor of Nitrogenase Binds and Reduces
Atmospheric Nitrogen
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. The reductase (also called the iron protein or the Fe
protein) is a dimer of identical 30-kd subunits bridged by a 4Fe-4S cluster (Figure 24.3).
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.
The nitrogenase component is an α 2 β 2 tetramer (240 kd), in which the α and β subunits are homologous to each other
and structurally quite similar (Figure 24.4). 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 molybdenumcontaining enzymes except nitrogenase.
The FeMo cofactor is the site of nitrogen fixation. Note that each of the six central iron atoms is linked to only three
atoms, leaving open a binding opportunity for N2. It seems likely that N2 binds in the central cavity of this cofactor
(Figure 24.5). The formation of multiple Fe-N interactions in this complex weakens the N N bond and thereby lowers
the activation barrier for reduction.
24.1.2. Ammonium Ion Is Assimilated into an Amino Acid Through Glutamate and
Glutamine
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.
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 24.6). 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 NH
+
4
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 high affinity for NH4 +. Thus, ATP hydrolysis is required to capture ammonia when it is
scarce.
III. Synthesizing the Molecules of Life
24. The Biosynthesis of Amino Acids
24.1. Nitrogen Fixation: Microorganisms Use ATP and a Powerful Reductant to Reduce Atmospheric Nitrogen to Ammonia
Figure 24.2. Nitrogen Fixation. 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.
III. Synthesizing the Molecules of Life
24. The Biosynthesis of Amino Acids
24.1. Nitrogen Fixation: Microorganisms Use ATP and a Powerful Reductant to Reduce Atmospheric Nitrogen to Ammonia
Figure 24.3. Fe Protein. 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.
III. Synthesizing the Molecules of Life
24. The Biosynthesis of Amino Acids
24.1. Nitrogen Fixation: Microorganisms Use ATP and a Powerful Reductant to Reduce Atmospheric Nitrogen to Ammonia
Figure 24.4. MOFe Protein. 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.
III. Synthesizing the Molecules of Life
24. The Biosynthesis of Amino Acids
24.1. Nitrogen Fixation: Microorganisms Use ATP and a Powerful Reductant to Reduce Atmospheric Nitrogen to Ammonia
Figure 24.5. Nitrogen-Reduction Site. The FeMo cofactor contains an open center that is the likely site of nitrogen
binding and reduction.
III. Synthesizing the Molecules of Life
24. The Biosynthesis of Amino Acids
24.1. Nitrogen Fixation: Microorganisms Use ATP and a Powerful Reductant to Reduce Atmospheric Nitrogen to Ammonia