22.1 Overview of Nitrogen Metabolism in Chapter 22 Biosynthesis of Amino Acids, Nucleotides, and Related Molecules

22.1 Overview of Nitrogen Metabolism

The biosynthetic pathways leading to amino acids and nucleotides share a requirement for nitrogen. Soluble, biologically useful nitrogen compounds are generally scarce in natural environments; thus, most organisms use ammonia, amino acids, and nucleotides economically. Available amino acids, purines, and pyrimidines formed during metabolic turnover of proteins and nucleic acids are often salvaged and reused. We begin by examining the pathways by which nitrogen from the environment is introduced into biological systems.

A Global Nitrogen Cycling Network Maintains a Pool of Biologically Available Nitrogen

The movement of nitrogen through the biosphere has been viewed historically as a cycle. However, our evolving understanding of the complexity of nitrogenous interconversions makes it clear that nitrogen moves through a complex web, rather than in a neat cycle (Fig. 22-1). Earth’s atmosphere is four-fifths molecular nitrogen $(N_{2})$ $left-parenthesis upper N Subscript 2 Baseline right-parenthesis$ . However, $N_{2}$ $upper N Subscript 2$ is too unreactive to be of use to living organisms. Conversion of $N_{2}$ $upper N Subscript 2$ to forms that can support life $({NH}_{3}, {NO}_{2}^{-}, {NO}_{3}^{-})$ $left-parenthesis NH Subscript 3 Baseline comma NO Subscript 2 Superscript minus Baseline comma NO Subscript 3 Superscript minus Baseline right-parenthesis$ is called nitrogen fixation. The reduction of $N_{2}$ $upper N Subscript 2$ to ${NH}_{3}$ $NH Subscript 3$ plays such a central role in making $N_{2}$ $upper N Subscript 2$ available that this one reaction is often considered synonymous with nitrogen fixation. In the biosphere, the metabolic processes of countless species function interdependently to salvage and reuse biologically available nitrogen. Most of the key reactions are carried out by bacteria and archaea.

A figure shows the parts of the global nitrogen web, illustrating how nitrogen moves among compounds and the corresponding oxidation numbers. — FIGURE 22-1 The global nitrogen web. The total amount of nitrogen fixed annually in the biosphere exceeds $10^{11} kg$ $10 Superscript 11 Baseline kg$ ; industrial sources of fixed nitrogen are now nearly as great. Reactions are identified with the processes they are involved in by colored arrows (see key). The oxidation number of the N atom is indicated on the vertical axis. [Information from M. M. M. Kuypers et al., *Nat. Rev. Microbiol.* 16:263, 2018, Fig. 1.]

FIGURE 22-1 The global nitrogen web. The total amount of nitrogen fixed annually in the biosphere exceeds $10^{11} kg$ $10 Superscript 11 Baseline kg$ ; industrial sources of fixed nitrogen are now nearly as great. Reactions are identified with the processes they are involved in by colored arrows (see key). The oxidation number of the N atom is indicated on the vertical axis. [Information from M. M. M. Kuypers et al., *Nat. Rev. Microbiol.* 16:263, 2018, Fig. 1.]

On the left side of the figure, an upward-pointing arrow is labeled oxidation and a downward-pointing arrow is labeled reduction. A vertical scale in the middle ranges from negative 3 to 5, labeled in increments of 1. The legend indicates that upward-pointing arrow symbols represent processes as follows: black equals nitrogen fixation, purple equals nitrification, red equals denitrification, blue equals anammox, and green equals assimilation. The following compounds are shown in the figure with their corresponding oxidation numbers: Ammonia: negative 3, N H 3; Dinitrogen gas: 0, N 2; Nitrous oxide: 1; N 2 O; Nitric oxide: 2, N O; Nitrite: 3, N O 2 minus; Nitrate: 5, N O 3 minus. The following reactions are shown. Organic nitrogen is shown at the bottom center with upward- and downward-pointing arrows extending to N H 3 above, indicating a reversible reaction. A purple arrow points from N H 3 with an oxidation number of negative 3 to N O with an oxidation number of 2, from which a purple arrow points to N O 2 minus with an oxidation number of 3, from which a purple arrow points to N O 3 minus with an oxidation number of 5. A green arrow points down from N O 3 minus to N O 2 minus, from which a green arrow points down to N H 3. A red arrow points down from N O 3 minus to N O 2 minus, from which a red arrow points down to N O, from which a red arrow points down to N 2 O with a dashed arrow pointing left to N 2 O, from which a red arrow points down to N 2. A blue arrow points down from N O 2 minus to N 2, from which a blue arrow points down to N H 3. A black arrow points down from N 2 to N H 3.

The reduction of atmospheric nitrogen $(N_{2})$ $left-parenthesis upper N Subscript 2 Baseline right-parenthesis$ by nitrogen-fixing bacteria and archaea to yield ammonia $({NH}_{3} or {NH}_{4}^{+})$ $left-parenthesis NH Subscript 3 Baseline or NH Subscript 4 Superscript plus Baseline right-parenthesis$ , provides a useful anchor for our discussion. This critical process, described in detail in Section 22.2, provides most of the reduced nitrogen for incorporation into biomolecules.

Free ammonia does not build up, and reduction is balanced by oxidation. Bacteria that derive their energy by oxidizing ammonia to nitrite $({NO}_{2}^{-})$ $left-parenthesis NO Subscript 2 Superscript minus Baseline right-parenthesis$ and ultimately nitrate $({NO}_{3}^{-})$ $left-parenthesis NO Subscript 3 Superscript minus Baseline right-parenthesis$ are abundant and active in both terrestrial and marine environments. The processes of converting ammonia to nitric oxide, nitrite, and finally nitrate are known as nitrification (Fig. 22-1, purple arrows). Individual bacterial or archaeal species may promote one or more of these steps.

Atmospheric $N_{2}$ $upper N Subscript 2$ must be replaced in order to maintain levels at a steady-state concentration. Some of the replacement comes from reduction of nitrate and nitrite. The reduction of nitrate and nitrite to $N_{2}$ $upper N Subscript 2$ under anaerobic conditions, a process called denitrification (Fig. 22-1, red arrows), is carried out by specialized microorganisms in all three domains of life. These organisms use ${NO}_{3}^{-}$ $NO Subscript 3 Superscript minus$ or ${NO}_{2}^{-}$ $NO Subscript 2 Superscript minus$ rather than $O_{2}$ $upper O Subscript 2$ as the ultimate electron acceptor in a series of reactions that (like oxidative phosphorylation) generates a transmembrane proton gradient, which is used to synthesize ATP. These microorganisms exist in all anoxic environments where nitrate is present, including soils, marine sediments, and eutrophic marine zones. An alternative path back to atmospheric $N_{2}$ $upper N Subscript 2$ is provided by a group of bacteria that promote anaerobic ammonia oxidation, or anammox (Fig. 22-1, blue arrows). Anammox converts ammonia and nitrite to $N_{2}$ $upper N Subscript 2$ . As much as 50% to 70% of the ${NH}_{3} {-to-N}_{2}$ $NH Subscript 3 Baseline hyphen to hyphen upper N Subscript 2$ conversion in the biosphere may occur through this pathway, which went undetected until the 1980s. The obligate anaerobes that promote anammox are fascinating in their own right and are providing some useful solutions to waste-treatment problems (Box 22-1).

BOX 22-1 Unusual Lifestyles of the Obscure but Abundant

Air-breathers that we are, we can easily overlook the bacteria and archaea that thrive in anaerobic environments. Although rarely featured in introductory biochemistry textbooks, these organisms constitute much of the biomass of this planet, and their contributions to the balance of carbon and nitrogen in the biosphere are essential to all forms of life.

As detailed in earlier chapters, the energy used to maintain living systems relies on the generation of proton gradients across membranes. Electrons derived from a reduced substrate are made available to electron carriers in membranes and pass through a series of electron transfers to a final electron acceptor. As a byproduct of this process, protons are released on one side of the membrane, generating the transmembrane proton gradient. The proton gradient is used to synthesize ATP or to drive other energy-requiring processes. For all eukaryotes, the reduced substrate is generally a carbohydrate (glucose or pyruvate) or a fatty acid and the electron acceptor is oxygen.

Many bacteria and archaea are much more versatile. In anaerobic environments such as marine and freshwater sediments, the variety of life strategies is extraordinary. Almost any available redox pair can be an energy source for some specialized organism or group of organisms. For example, a large number of lithotrophic bacteria (a lithotroph is a chemotroph that uses inorganic energy sources) have a hydrogenase that uses molecular hydrogen to reduce ${NAD}^{+}$ $NAD Superscript plus$ :

H_{2} + {NAD}^{+} \overset{hydrogenase}{\to} NADH + H^{+}

$upper H Subscript 2 Baseline plus NAD Superscript plus Baseline right-arrow Overscript hydrogenase Endscripts NADH plus upper H Superscript plus$

The NADH is a source of electrons for a variety of membrane-bound electron acceptors, generating the proton gradient needed for ATP synthesis. Other lithotrophs oxidize sulfur compounds ( $H_{2} S$ $upper H Subscript 2 Baseline upper S$ , elemental sulfur, or thiosulfate) or ferrous iron. A widespread group of archaea called methanogens, all strict anaerobes, extract energy from the reduction of ${CO}_{2}$ $CO Subscript 2$ to methane. And this is just a small sampling of what anaerobic organisms do for a living. Their metabolic pathways are replete with interesting reactions and highly specialized cofactors unknown in our own world of obligate aerobic metabolism. Study of these organisms can yield practical dividends. It can also provide clues about the origins of life on an early Earth, in an atmosphere that lacked molecular oxygen.

The web of reactions that defines nitrogen utilization in the biosphere depends on a wide range of specialized bacteria. Some nitrifying bacteria oxidize ammonia to nitrites, and some oxidize the resulting nitrites to nitrates (see Fig. 22-1, purple arrows). Nitrate is second only to $O_{2}$ $upper O Subscript 2$ as a biological electron acceptor, and a great many bacteria and archaea can catalyze the denitrification of nitrates and nitrites to nitrogen, which the nitrogen-fixing bacteria then convert back into ammonia. Ammonia is a major pollutant in sewage and in farm animal waste, and it is a byproduct of fertilizer manufacture and oil refining. Waste-treatment plants have generally made use of communities of nitrifying and denitrifying bacteria to convert ammonia waste to atmospheric nitrogen. The process is expensive, requiring inputs of organic carbon and oxygen.

In the 1960s and 1970s, a few articles appeared in the research literature suggesting that ammonia could be oxidized to nitrogen anaerobically, using nitrite as an electron acceptor; this process was called anammox. The reports received little notice until bacteria promoting anammox were discovered in a waste-treatment system in Delft, the Netherlands, in the mid-1980s. A team of Dutch microbiologists led by Gijs Kuenen and Mike Jetten began to study these bacteria, which were soon identified as belonging to an unusual bacterial phylum, Planctomycetes. Some surprises were to follow.

The biochemistry underlying the anammox process was slowly unraveled (Fig. 1). Hydrazine $(N_{2} H_{4})$ $left-parenthesis upper N Subscript 2 Baseline upper H Subscript 4 Baseline right-parenthesis$ , a highly reactive molecule used as a rocket fuel, was an unexpected intermediate. As a small molecule, hydrazine is both highly toxic and difficult to contain. It readily diffuses across typical phospholipid membranes. The anammox bacteria solve this problem by sequestering hydrazine in a specialized organelle, dubbed the anammoxosome. The membrane of this organelle is composed of lipids known as ladderanes (Fig. 2), never before encountered in biology. The fused cyclobutane rings of ladderanes stack tightly to form a very dense barrier, greatly slowing the release of hydrazine. Cyclobutane rings, with their unusual bond angles, are strained and difficult to synthesize; the bacterial mechanisms for synthesizing these lipids are not yet known.

FIGURE 1 The anammox reactions. Ammonia and hydroxylamine are converted to hydrazine and $H_{2} O$ $upper H Subscript 2 Baseline upper O$ by hydrazine hydrolase, and the hydrazine is oxidized by hydrazine-oxidizing enzyme, generating $N_{2}$ $upper N Subscript 2$ and protons. The protons create a proton gradient for ATP synthesis. On the anammoxosome exterior, protons are used by the nitrite-reducing enzyme, producing hydroxylamine and completing the cycle. All of the anammox enzymes are embedded in the anammoxosome membrane. [Information from L. A. van Niftrik et al., FEMS Microbiol. Lett. 233:10, 2004, Fig. 4.]

A horizontal piece of membrane is shown with the region above labeled cytosol and the region below labeled anammoxosome. A gray rectangle embedded in the left side of the membrane is labeled hydroaxine hydrolase. A purple rectangle labeled nitrite-reducing enzyme is embedded into the cytosolic top side of the membrane to its right. A green rectangle labeled hydrazine-oxidizing enzyme is embedded in the bottom side of the membrane to its right. An A T P synthase at the far right has a cylindrical portion embedded in the membrane. This cylinder consists of many vertical cylindrical pieces smoothly joined together, rather than being separate cylinders, and is labeled C 10. A flattened bar to its left labeled a is rounded to fit against the cylinder. A thin green cylinder labeled gamma emerges from the center of the cylinder and a blue oval labeled epsilon is visible behind and slightly to the left of the thin green cylinder. The thin green cylinder runs up through a sphere made up of vertical ovals. Clockwise from a dark gray oval in front labeled gamma, these are a light purple oval labeled beta, a light gray oval labeled alpha, a purple oval labeled beta, a gray oval labeled alpha, and a dark purple oval labeled beta. A small orange sphere labeled delta is at the top of the thin green cylinder between the ovals and two joined strands labeled b subscript 2 end subscript run from is down the left side of the structure to end along the thin flattened bar labeled a. Arrows from N H 3 at the upper left and N H 2 O H to its right, which is produced by the nitrite-reducing enzyme, join as they meet the top of the membrane and point into the gray square labeled hydrazine hydrolase. An arrow emerges from the bottom of hydrazine hydrolase and splits to indicate H 2 O to the left and N 2 H 4 to the right, from which an arrow points to the green rectangle labeled hydrazine-oxidizing enzyme. Three arrows leave the hydrazine-oxidizing enzyme. One arrow points up to an orange oval labeled 4 e minus in the middle of the membrane, from which an arrow points into the purple rectangle labeled nitrite-reducing enzyme embedded in the top of the membrane. Additional arrows point into the nitrite-reducing enzyme from 5 H plus and N O 2 minus and arrows indicate that H 2 O and N H 2 O H are produced, with N H 2 O H continuing down to hydrazine hydrolase. A second arrow from the hydrazine-oxidizing enzyme points down to N 2 and the third points to red highlighted 4 H plus, from which a vertical arrow points up into the flattened bar of A T P synthase that is labeled a. This arrow curves right to indicate a series of four right-pointing arrows around the outside of the large cylinder at the base of A T P synthase, C subscript 10 end subscript. A black arrow beginning just past the blue oval labeled epsilon runs counter clockwise along the top of this cylinder until it reaches the left side of the same blue oval. A red arrow points from the main cylinder into the thin bar labeled a and points upward. A curved arrow showing the input of A D P plus P subscript i end subscript and production of A T P meets the dark purple oval of the rounded top of A T P synthase.

FIGURE 2 (a) Ladderane lipids of the anammoxosome membrane. The mechanism for synthesis of the unstable fused cyclobutane ring structures is unknown. (b) Ladderanes can stack to form a very dense, impermeable, hydrophobic membrane structure, allowing sequestration of the hydrazine produced in the anammox reactions. [Information from L. A. van Niftrik et al., FEMS Microbiol. Lett. 233:7, 2004, Fig. 3.]

Part a shows three examples of lipids. The top structure has C double bonded to O above and bonded to O minus to the left, all in a blue box, and further bonded to a seven-carbon zigzag chain to the left outside of the blue box that ends at the lower left vertex of a parallelogram that angles down to the right and shares its right side with the left side of the adjacent parallelogram that angles up to the right. These two parallelograms are the first in a series of five parallelograms that angle in alternate directions. Below, a vertical three-carbon chain in a light blue box has C 1 at the top bonded to 2 H and to O H on the left, C 2 bonded to H and to O on the right further bonded to an eight-carbon zigzag chain to the right outside of the blue box that ends at the lower left vertex of a parallelogram that is further bonded, and C 3 bonded to 2 H and to O H on the left. The parallelogram resembles the first two parallelograms of the molecule above, with half angling down and the other half angling up, but with the two halves joined. The remaining three parallelograms of the series have a similar pattern to the molecule above. The third molecule resembles the second molecule except that C 3 is bonded to 2 H and to O on the right further bonded to C double bonded to O below and bonded to a seven-carbon zigzag chain outside of the blue box that ends with a bond to the upper left vertex of a series of five parallelograms that begins with a parallelogram that angles up to the right but otherwise resembles the series of parallelograms in the other molecules with alternating angles of the parallelograms. Part b shows a vertical series of nine blue spheres with each separated from the next by a smaller blue sphere. All of the blue spheres, small and large, are bonded to eight-carbon zigzag chains that fit tightly together and each zigzag chain ends with a series of parallelograms. Each series of parallelograms is adjacent to the series of parallelograms beneath. To the right, a similar set of structures is shown but with the parallelograms on the left, the zigzag chains in the middle, with the blue spheres on the right, and with a small blue sphere bonded to the top chain instead of a large blue sphere bonded to the top chain.

The anammoxosome was a surprising finding. Bacterial cells generally do not have compartments, and the lack of a membrane-enclosed nucleus is often cited as the primary distinction between eukaryotes and bacteria. One type of organelle in a bacterium was interesting enough, but microbiologists also found that planctomycetes have a nucleus: their chromosomal DNA is contained within a membrane (Fig. 3). Planctomycetes are an ancient bacterial line with multiple genera, three of which are known to carry out the anammox reactions. Discovery of this subcellular organization has prompted further research to trace the origin of the planctomycetes and the evolution of eukaryotic nuclei. Further study of this group may ultimately bring us closer to a key goal of evolutionary biology: a description of the organism affectionately referred to as LUCA — the last universal common ancestor of all life on our planet.

FIGURE 3 Transmission electron micrograph of a cross section through Gemmata obscuriglobus, showing the DNA in a nucleus (N) with enclosing nuclear envelope (NE). Bacteria of the Gemmata genus (phylum Planctomycetes) do not promote the anammox reactions.

For now, the anammox bacteria offer a major advance in waste treatment, reducing the cost of ammonia removal by as much as 90% (the conventional denitrification steps are eliminated completely, and the aeration costs associated with nitrification are lower) and reducing the release of polluting byproducts. Clearly, a greater familiarity with the bacterial underpinnings of the biosphere can pay big dividends as we deal with the environmental challenges of the twenty-first century.

Fixation of atmospheric $N_{2}$ $upper N Subscript 2$ is not the only source of reduced ammonia for biological systems. Much of it comes from an alternative fate of nitrate that circumvents denitrification. More than 90% of the ${NH}_{4}^{+}$ $NH Subscript 4 Superscript plus$ generated by vascular plants, algae, and microorganisms comes from nitrate assimilation, a two-step reductive process that bypasses atmospheric $N_{2}$ $upper N Subscript 2$ . First ${NO}_{3}^{-}$ $NO Subscript 3 Superscript minus$ is reduced to ${NO}_{2}^{-}$ $NO Subscript 2 Superscript minus$ by nitrate reductase, then the ${NO}_{2}^{-}$ $NO Subscript 2 Superscript minus$ is reduced to ${NH}_{4}^{+}$ $NH Subscript 4 Superscript plus$ in a six-electron transfer catalyzed by nitrite reductase (Fig. 22-2). Both reactions involve chains of electron carriers and cofactors we have not yet encountered. Nitrate reductase is a large, soluble protein $(M_{r} 220, 000)$ $left-parenthesis upper M Subscript r Baseline 220 comma 000 right-parenthesis$ . Within the enzyme, a pair of electrons, donated by NADH, flows through $— SH$ $em-dash SH$ groups of cysteine, FAD, and a cytochrome $(cyt b_{557})$ $left-parenthesis cyt b 557 right-parenthesis$ , then to a novel cofactor containing molybdenum, before reducing the substrate ${NO}_{3}^{-}$ $NO Subscript 3 Superscript minus$ to ${NO}_{2}^{-}$ $NO Subscript 2 Superscript minus$ .

A two-part figure shows nitrate assimilation by nitrate reductase in two stages: a step in which N O 3 minus is converted to N O 2 minus that involves the use of a Mo-containing cofactor in part a and a step in which N O 2 minus is converted to N H 4 plus in a proton-transfer process that involves the metallic center in siroheme. — FIGURE 22-2 Nitrate assimilation by nitrate reductase and nitrite reductase. (a) Nitrate reductases of plants and bacteria catalyze the two-electron reduction of ${NO}_{3}^{-}$ $NO Subscript 3 Superscript minus$ to ${NO}_{2}^{-}$ $NO Subscript 2 Superscript minus$ , in which a novel Mo-containing cofactor plays a central role. NADH is the electron donor. (b) Nitrite reductase converts the product of nitrate reductase into ${NH}_{4}^{+}$ $NH Subscript 4 Superscript plus$ in a six-electron, eight-proton transfer process in which the metallic center in siroheme carries electrons and the carboxyl groups of siroheme may donate protons. The initial source of electrons is reduced ferredoxin.

FIGURE 22-2 Nitrate assimilation by nitrate reductase and nitrite reductase. (a) Nitrate reductases of plants and bacteria catalyze the two-electron reduction of ${NO}_{3}^{-}$ $NO Subscript 3 Superscript minus$ to ${NO}_{2}^{-}$ $NO Subscript 2 Superscript minus$ , in which a novel Mo-containing cofactor plays a central role. NADH is the electron donor. (b) Nitrite reductase converts the product of nitrate reductase into ${NH}_{4}^{+}$ $NH Subscript 4 Superscript plus$ in a six-electron, eight-proton transfer process in which the metallic center in siroheme carries electrons and the carboxyl groups of siroheme may donate protons. The initial source of electrons is reduced ferredoxin.

The vertical reaction runs from top to bottom across parts a and b as follows: N O 3 minus is shown at the top center. An arrow points down from N O 3 minus to N O 2 minus below accompanied by a curved line showing the addition of 2 e minus from a reaction shown in part a. An arrow points down from N O 2 minus to N H 4 plus at the bottom center accompanied by a curved line showing the addition of 6 e minus from the reaction shown in part b. Part a shows a blue rectangle with rounded corners labeled nitrate reductase. Within this rectangle, an arrow points right from S H further bonded to the left to F A D, from which an arrow points right to C y t italicized b end italics subscript 557 end subscript, from which an arrow points down to Mo Co, from which an arrow points right to the 2 e minus that joins the arrow pointing down from N O 3 minus to N O 2 minus. A curved arrow from N A D H to N A D plus meets the blue rectangle on its left side. To the right, the Mo cofactor is shown as a horizontal three-ring structure. The left-hand ring has double bonds at its lower left side and at its right side shared with the central ring, N substituted for C at the bottom vertex, a lower left vertex bonded to N H 2, N H substituted for C at the upper left vertex, and a top vertex double bonded to O. The central ring has a double bond at its left side shared with the left-hand ring, a single right side bond shared with the right-hand ring, and N H substituted for C at the top and bottom vertices. The right-hand ring shares its left side with the central ring, has O substituted for C at its bottom vertex, and has a double bond at its upper right side. The double bond at its upper right vertex forms the angled base of a five-membered ring with S substituted for C at the two side vertices and with M o in a blue circle at the top vertex and further double bonded to O to the upper left and right. Additionally, the lower right vertex of the right-hand ring is bonded to C H 2 bonded to O bonded to P to the lower right that is bonded to O minus to the upper and lower left and double bonded to O to the lower right. Part b shows a blue oval labeled nitrate reductase. Within the blue oval, an arrow points right from F e bonded to S to siroheme, from which an arrow points right to 6 e minus that joins the arrow pointing down from N O 2 minus to N H 4 plus. A curved arrow that extends from a gray oval labeled F d subscript red end subscript to an orange oval labeled F d subscript ox end subscript meets the blue oval at its left side. Siroheme is shown with an orange circle labeled F e 2 plus in the center surrounded by four five-membered rings around the four outer corners with four partial six-membered rings in between them. The top left five-membered ring has a double bond at the right side shared with the left side of the six-membered top central ring, a bottom bond shared with the top bond of the partial six-membered ring below, N substituted for C at the lower right vertex with a red arrow pointing to the central F e 2, an upper left vertex hashed wedge bonded to C H 3 and solid wedge bonded to C H 2 bonded to C O O H, and a top vertex hashed wedge bonded to C H 3 bonded to C H 2 bonded to C O O H. The left center partial six-membered ring is expanded to the right, where it has N at its upper and lower right vertices each with red arrows pointing to F e 2 plus in the center that serve as the upper and lower right sides, respectively. It has a top side shared with the five-membered ring above, a bottom side shared with the five-membered ring below, and a double bond at the upper left side. The lower left five-membered ring shares its top side with the partial six-membered ring above, has N at the upper right vertex shared with the partial six-membered rings above and to the right, has double bonds at its upper left and lower right sides, has a lower left vertex bonded to C H 2 bonded to C O O H, and has a bottom vertex bonded to C H 2 bonded to C H 2 bonded to C O O H. The partial six-membered ring at the bottom center is elongated at its top where the N atoms shared with the surrounding rings and located at its upper left and right vertices are each connected by red arrows to F e 2 plus in the center to form the upper left and upper right sides, respectively. There is a double bond at the lower left side and the right-side is shared with the five-membered ring to its right. The lower right five-membered ring has a left side shared with the partial six-membered ring to its left, N at its upper left vertex shared with the partial six-membered rings to the left and above that has a red arrow to F e 2 plus in the center, has a double bond at its lower right side and a double bond at its top side shared with the partial six-membered ring above, has a bottom vertex bonded to C H 2 bonded to C O H 2 bonded to C O O H, and has a lower right side vertex bonded to C H 2 bonded to C O O H. The partial six-membered ring at the center right is elongated to the left, where it has shared N atoms at the upper and lower left vertices that each have red arrows pointing to F e 2 plus in the center that form the upper and lower right sides, respectively. It has a top side shared with the five-membered ring above, a double bond at it supper right side, and a double bond at its bottom side shared with the five-membered ring below. The five-membered ring at the upper right has a bottom bond shared with the partial six-membered ring below, a left side bond shared with the partial six-membered ring to its left, a shared N at its lower left vertex with a red arrow pointing to F e 2 plus in the center, a right side vertex hashed wedge bonded to C H 2 boned to C H 2 bonded to C O O H, and a top vertex hashed wedge bonded to C H 3 and solid wedge bonded to C H 2 bonded to C O O H. The top center partial six-membered ring is elongated at its bottom where N atoms at its lower left and right vertices each have red arrows pointing to F e 2 plus in the enter that form the left and right sides of the ring, respectively. It shares its right side with the five-membered ring to the right and a double bond at its left side with the five-membered ring to its left. It also has a double bond at its upper right side.

The nitrite reductase of plants is located in the chloroplasts and receives its electrons from ferredoxin (which is reduced in the light-dependent reactions of photosynthesis; see Section 20.2). Six electrons, donated one at a time by ferredoxin, pass through a 4Fe-4S center in the enzyme, then through a novel hemelike molecule (siroheme) before reducing ${NO}_{2}^{-}$ $NO Subscript 2 Superscript minus$ to ${NH}_{4}^{+}$ $NH Subscript 4 Superscript plus$ (Fig. 22-2). Nonphotosynthetic microbes possess a distinct nitrite reductase for which NADPH is the electron donor.

Human activity presents an increasing challenge to the global nitrogen balance, and to all life in the biosphere supported by that balance. Fixed nitrogen is increasingly necessary to boost production in agriculture. Industrial nitrogen-based fertilizers now contribute as much ammonia and other reactive nitrogen species to the biosphere as do natural processes. Nonfarming manufacturing activity releases additional reactive nitrogen into the atmosphere, including nitric oxide, a prominent greenhouse gas. Controlling the damaging effects of agricultural runoff and industrial pollutants will remain an important component of the continuing effort to expand the food supply for a growing human population.

Nitrogen Is Fixed by Enzymes of the Nitrogenase Complex

The availability of fixed nitrogen, an essential nutrient, may have limited the size of the primordial biosphere. As early cells acquired a capacity to fix atmospheric nitrogen, the biosphere expanded. Evidence for biological nitrogen fixation has been found in sedimentary rocks more than 3 billion years old.

In the biosphere of today, only certain bacteria and archaea can fix atmospheric $N_{2}$ $upper N Subscript 2$ . These organisms, called diazotrophs, include the cyanobacteria of soils and fresh and salt waters, methanogenic archaea (strict anaerobes that obtain energy and carbon by converting $H_{2}$ $upper H Subscript 2$ and ${CO}_{2}$ $CO Subscript 2$ to methane), other kinds of free-living soil bacteria such as Azotobacter species, and the nitrogen-fixing bacteria that live as symbionts in the root nodules of leguminous plants. The most important product of nitrogen fixation is ammonia, which can be used by all organisms either directly or after its conversion to other soluble compounds such as nitrites, nitrates, or amino acids.

The reduction of nitrogen to ammonia is an exergonic reaction:

\begin{matrix} N_{2} + 3 H_{2} \to 2 {NH}_{3} & Δ G^{'} ° = − 33.5 kJ/mol \end{matrix}

$StartLayout 1st Row 1st Column upper N Subscript 2 Baseline plus 3 upper H Subscript 2 Baseline right-arrow 2 NH Subscript 3 Baseline 2nd Column Blank 3rd Column upper Delta upper G Superscript prime Baseline degree equals negative 33.5 kJ slash mol EndLayout$

The $N ≡ N$ $upper N identical-to upper N$ triple bond, however, is very stable, with a bond energy of 930 kJ/mol. Nitrogen fixation therefore has an extremely high activation energy, and atmospheric nitrogen is almost chemically inert under normal conditions. Ammonia is produced industrially by the Haber process (named for its inventor, Fritz Haber), which requires temperatures of 400 to 500 °C and nitrogen and hydrogen at pressures of tens of thousands of kilopascals (several hundred atmospheres) to provide the necessary activation energy.

Biological nitrogen fixation must occur at biological temperatures and at 0.8 atm of nitrogen, and the high activation barrier is overcome by other means. This is accomplished, at least in part, by the binding and hydrolysis of ATP. The overall reaction can be written

N_{2} + 10 H^{+} + 8 e^{-} + 16 ATP \to 2 {NH}_{4}^{+} + 16 ADP + 16 P_{i} + H_{2}

$upper N Subscript 2 Baseline plus 10 upper H Superscript plus Baseline plus 8 e Superscript en-dash Baseline plus 16 ATP right-arrow 2 NH Subscript 4 Superscript plus Baseline plus 16 ADP plus 16 upper P Subscript i Baseline plus upper H Subscript 2 Baseline$

Biological nitrogen fixation to produce ammonia is carried out by a highly conserved complex of proteins called the nitrogenase complex; its central components are dinitrogenase reductase and dinitrogenase (Fig. 22-3a). Dinitrogenase reductase $(M_{r} 60, 000)$ $left-parenthesis upper M Subscript r Baseline 60 comma 000 right-parenthesis$ is a dimer of two identical subunits. It contains a single 4Fe-4S redox center (see Fig. 19-5), bound between the subunits, and can be oxidized and reduced by one electron. It also has two binding sites for ATP/ADP (one site on each subunit). Dinitrogenase $(M_{r} 240, 000)$ $left-parenthesis upper M Subscript r Baseline 240 comma 000 right-parenthesis$ , an $α_{2} β_{2}$ $alpha 2 beta 2$ tetramer, has two Fe-containing cofactors that transfer electrons (Fig. 22-3b). One, the P cluster, has a pair of 4Fe-4S centers; these share a sulfur atom, making an 8Fe-7S center. The second cofactor in dinitrogenase, the FeMo cofactor, is a novel structure composed of 7 Fe atoms, 9 inorganic S atoms, a Cys side chain, and a single carbon atom in the center of the FeS cluster. Also part of the cofactor is a molybdenum atom, with ligands that include three inorganic S atoms, a His side chain, and two oxygen atoms from a molecule of homocitrate that is an intrinsic part of the FeMo cofactor.

A two-part figure shows the holoenzyme consisting of two dinitrogenase reductase molecules that make up the nitrogenase complex in part a and the structures of the electron-transfer cofactors in part b. — FIGURE 22-3 Enzymes and cofactors of the nitrogenase complex. (a) The holoenzyme consists of two identical dinitrogenase reductase molecules (green), each with a 4Fe-4S redox center and binding sites for two ATP, and two identical dinitrogenase heterodimers (purple and blue), each with a P cluster (Fe-S center) and an FeMo cofactor. In this structure, ADP is bound in the ATP site, to make the crystal more stable. (b) The electron-transfer cofactors. A P cluster is shown here in its reduced (top) and oxidized (middle) forms. The FeMo cofactor (bottom) has a Mo atom with three S ligands, a His ligand, and two oxygen ligands from a molecule of homocitrate. In some organisms, the Mo atom is replaced with a vanadium atom. (Fe is shown in orange, S in yellow.) [Data from (a) PDB ID 1N2C, H. Schindelin et al., *Nature* 387:370, 1997; (b) $P_{red}$ $upper P Subscript red$ : PDB ID 3MIN, and $P_{ox}$ $upper P Subscript ox$ : PDB ID 2MIN, J. W. Peters et al., *Biochemistry* 36:1181, 1997; FeMo cofactor: PDB ID 1M1N, O. Einsle et al., *Science* 297:1696, 2002.]

FIGURE 22-3 Enzymes and cofactors of the nitrogenase complex. (a) The holoenzyme consists of two identical dinitrogenase reductase molecules (green), each with a 4Fe-4S redox center and binding sites for two ATP, and two identical dinitrogenase heterodimers (purple and blue), each with a P cluster (Fe-S center) and an FeMo cofactor. In this structure, ADP is bound in the ATP site, to make the crystal more stable. (b) The electron-transfer cofactors. A P cluster is shown here in its reduced (top) and oxidized (middle) forms. The FeMo cofactor (bottom) has a Mo atom with three S ligands, a His ligand, and two oxygen ligands from a molecule of homocitrate. In some organisms, the Mo atom is replaced with a vanadium atom. (Fe is shown in orange, S in yellow.) [Data from (a) PDB ID 1N2C, H. Schindelin et al., *Nature* 387:370, 1997; (b) $P_{red}$ $upper P Subscript red$ : PDB ID 3MIN, and $P_{ox}$ $upper P Subscript ox$ : PDB ID 2MIN, J. W. Peters et al., *Biochemistry* 36:1181, 1997; FeMo cofactor: PDB ID 1M1N, O. Einsle et al., *Science* 297:1696, 2002.]

Part a shows a vertical structure with a green roughly rectangular structure at the top above two blue roughly rectangular structures above a similar green roughly rectangular structure. There is a small opening between the top blue structures. To the left of the top blue structure, there is a roughly rounded purple structure. A similar structure is to the right of the bottom blue structure. The green structures are labeled dinitrogenase reductase. The blue structures represent dinitrogenase (beta subunit), and the purple structures represent dinitrogenase (alpha subunit). Near the top center of the top green structure, there are two almost parallel vertical space-filling models that each have a green sphere at the bottom, then a region of red and yellow, then a gray region with some red around the outside beneath a region of blue and black spheres. Near the bottom of the top green structure, there is a sphere consisting of red and orange spheres. At the top left of the top blue structure, there is a linear structure consisting of red and orange spheres. A tiny piece of pink is visible at the right between the upper blue and green structures. A large roughly rectangular space filling model that extends from the left side of the upper blue structure into the pink structure to its left contains gray spheres on the right with red spheres around them and a left side that consists of yellow and red spheres. Similar structures are visible in the lower green, blue, and purple structures but are inverted so that the two almost parallel vertical space-filling models are near the bottom of the green structure, the orange and yellow sphere is near the top of the green structure, the linear red and orange structure is near the bottom of the blue subunit, and the almost rectangular structure is at the right edge of the blue subunit extending into the right-hand purple subunit. A rectangle encompassing much of the top green structure, the upper part of the top blue structure, and the upper right corner of the upper purple structure is shown in close-up to the right. Two almost vertical ball-and-stick models of A D P are shown, each with a green M g beneath. An arrow points down to a cuboidal 4 F e – 4 S redox center, which is a cuboidal structure with yellow spheres at the upper left and right vertices and lower front and rear vertices and with orange spheres at the upper front and rear vertices and at the lower left and right vertices. An arrow labeled 6 e minus points down to a P cluster, shown as two adjacent cuboidal structures similar to the 4 F e – F S redox center. It has a cuboidal portion on the left and the upper right rear yellow sphere is bonded to two orange spheres at the upper left and right and an orange sphere at the rear lower left of the right-hand structure. Each of the orange spheres at the top is bonded to a yellow sphere at the upper right front vertex, completing the top of the structure, and to two yellow spheres below that each join to an orange sphere at the lower right front vertex that connects to the yellow sphere at the upper right front vertex above. An arrow labeled 6 e minus points down to the F e Mo cofactor, which is similar to the preceding structure except that a lower yellow vertex of the cube is adjacent to a red sphere bonded to a central black sphere in a chain of seven black spheres in which some black spheres have visible bonds to red spheres. An arrow labeled 6 e minus joins with an arrow from N 2 to yield 2 N H 3. Part b shows the detailed structures of the reduced form of the A P cluster, the oxidized form of the P cluster, and the F e Mo cofactor. The top structure, representing the reduced form of the P cluster, is a ball-and stick model labeled P subscript red end subscript. It has a symmetrical appearance that is almost rectangular in the middle with protrusions to the left, front, and right that end in black spheres and a protrusion above that ends at a blue sphere next to text reading, alpha-C y s superscript 88 end superscript. The left-and right-hand protrusions are roughly cuboidal protruding toward the center. It has a central yellow sphere bonded to red spheres to the upper left, upper right, front lower right, and front lower left. It has two longer bonds across the rear of the structure to red spheres to the left and right. The red spheres at the upper left and right rear are each bonded to the same yellow sphere above that is further bonded to a chain of two black spheres that ends at a blue sphere next to the labeled alpha-C y s superscript 88 end superscript. The red spheres at the upper left and right rear are also bonded to yellow spheres in the front that are bonded below to the lower left and right front. The red spheres at the left and right front are each bonded to the same orange sphere in the front center that is further bonded to a black sphere toward the viewer. The left-hand orange sphere forms the lower right vertex of a cube-like shape with yellow at the lower left front vertex, red at the lower left bottom vertex, the central yellow sphere to the lower right distorting the cube in that direction, a red sphere at the upper right rear, a yellow sphere at the upper left rear, and an orange sphere at the upper left front that is further bonded to a yellow sphere bonded to a black sphere. The red sphere at the lower left rear vertex is further bonded to a yellow sphere below to the rear that is further bonded to a black sphere. The right side is similar but in the opposite orientation, with the central yellow sphere forming the lower left rear vertex of the cube. P subscript ox end subscript has a similar configuration except that the center has opened up more. The yellow sphere at the top center is still bonded to a chain of two black spheres followed by a blue sphere, but the blue sphere is now bonded to the red sphere at the upper right rear vertex of the left-hand cube. This pulls the cube to the left. The central yellow sphere has been pulled toward the right, making the right-hand cube more cuboidal. The bottom structure is labeled F e Mo cofactor. It has a central structure with a central ring with a black sphere at the back, red to each side, and yellow in front. The rear black sphere is bonded to red spheres to the upper left and right that are each bonded to a yellow sphere below. The same upper left and right red spheres are each bonded to yellow spheres to the front that are bonded back to the red spheres of the ring below. The left-hand yellow sphere is bonded to a red sphere bonded to a yellow sphere below that is bonded to a red sphere behind and the left-hand red sphere of the central ring above. The red sphere behind is bonded to a yellow sphere below also bonded to a symmetrical red sphere to its upper right that is bonded to a red sphere to the right front. This yellow sphere forms the bottom of a diamond with yellow at the top and a green M o at the right side. The top yellow sphere of the diamond is bonded to a red sphere behind that is the right side of a rear diamond with yellow above, red to the left, and black at the rear vertex of the central ring below. The left side of the ring has a similar diamond with the left side red sphere of the central ring as its right vertex, yellow above and below, and red at the rear center. A ring labeled alpha-H i s superscript 195 end superscript is just to its left and consists of a black sphere bonded to a black sphere at the vertex of a ring that, counterclockwise from this black sphere, is blue, black, blue adjacent to the diamond to the right, and black. The blue Mo sphere at the right end joins to red spheres to the rear and front right. The rear red sphere bonds to a black sphere bonded to red above and to a chain of two black spheres that end with bonds to red spheres above and below in the front. The front red sphere bonds to a chain of black spheres that ends with a black sphere bonded to two red spheres.

There are two additional forms of nitrogenase. One includes a dinitrogenase with a vanadium-containing cofactor rather than molybdenum (VFe); the other contains a second Fe atom (FeFe). Each of the nitrogenase complexes is encoded by a separate set of genes. The FeMo nitrogenase complex is the ancestral type, and all nitrogen-fixing bacteria and archaea contain it. Some species can produce one or both of the alternative VFe or FeFe types. Although the alternative enzymes are somewhat less efficient, they may play important roles in environments in which molybdenum is limiting or absent. They may also permit some additional reactions to occur. The vanadium nitrogenase system of Azotobacter vinelandii has the remarkable capacity to catalyze the reduction of carbon monoxide (CO) to ethylene $(C_{2} H_{4})$ $left-parenthesis upper C Subscript 2 Baseline upper H Subscript 4 Baseline right-parenthesis$ , ethane, and propane.

Nitrogen fixation to produce ammonia is carried out by a highly reduced form of dinitrogenase and requires eight electrons: six for the reduction of $N_{2}$ $upper N Subscript 2$ and two to produce one molecule of $H_{2}$ $upper H Subscript 2$ . Production of $H_{2}$ $upper H Subscript 2$ is an obligate part of the reaction mechanism, but the biological role of $H_{2}$ $upper H Subscript 2$ in the process is not understood.

Dinitrogenase is reduced by the transfer of electrons from dinitrogenase reductase (Fig. 22-4). The dinitrogenase tetramer has two binding sites for the reductase. The required eight electrons are transferred from reductase to dinitrogenase one at a time: a reduced reductase molecule binds to the dinitrogenase and transfers a single electron, then the oxidized reductase dissociates from dinitrogenase, in a repeating cycle. Each turn of the cycle requires the hydrolysis of two ATP molecules by the dimeric reductase. The immediate source of electrons to reduce dinitrogenase reductase varies, with reduced ferredoxin (see Section 20.2), reduced flavodoxin, and perhaps other sources playing a role. In at least one species, the ultimate source of electrons to reduce ferredoxin is pyruvate.

A figure shows the path of electrons during nitrogen fixation by the nitrogenase complex. — FIGURE 22-4 Electron path in nitrogen fixation by the nitrogenase complex. Electrons are transferred from pyruvate to dinitrogenase via ferredoxin (or flavodoxin) and dinitrogenase reductase. Dinitrogenase reductase reduces dinitrogenase one electron at a time, with at least six electrons required to fix one molecule of $N_{2}$ $upper N Subscript 2$ . Two additional electrons are used to reduce $2 H^{+}$ $2 upper H Superscript plus$ to $H_{2}$ $upper H Subscript 2$ in a process that obligatorily accompanies nitrogen fixation in anaerobes, making a total of eight electrons required per $N_{2}$ $upper N Subscript 2$ molecule. The subunit structures and metal cofactors of the dinitrogenase reductase and dinitrogenase proteins are described in the text and in Figure 22-3.

FIGURE 22-4 Electron path in nitrogen fixation by the nitrogenase complex. Electrons are transferred from pyruvate to dinitrogenase via ferredoxin (or flavodoxin) and dinitrogenase reductase. Dinitrogenase reductase reduces dinitrogenase one electron at a time, with at least six electrons required to fix one molecule of $N_{2}$ $upper N Subscript 2$ . Two additional electrons are used to reduce $2 H^{+}$ $2 upper H Superscript plus$ to $H_{2}$ $upper H Subscript 2$ in a process that obligatorily accompanies nitrogen fixation in anaerobes, making a total of eight electrons required per $N_{2}$ $upper N Subscript 2$ molecule. The subunit structures and metal cofactors of the dinitrogenase reductase and dinitrogenase proteins are described in the text and in Figure 22-3.

A box at the upper left labeled 4 Co A plus 4 pyruvate has a curved arrow labeled 8 e minus to a box at the upper right labeled 4 C O 2 plus 4 acetyl-Co A that meets a curved arrow below from a gray oval labeled 8 ferredoxin or 8 flavodoxin (oxidized) on the left to an orange oval labeled 8 ferredoxin or 8 flavodoxin (reduced). The top arrow is red from the upper left until it meets the arrow below, after which it is gray and the arrow below is red until it reaches the orange oval. An arrow curving back from the orange oval labeled 8 ferredoxin or 8 flavodoxin (reduced) to the gray oval labeled labeled 8 ferredoxin or 8 flavodoxin (oxidized) is red until it meets an arrow below curving from a gray oval labeled 8 dinitrogenase reductase (oxidized) on the left that becomes red as it continues right to point to an orange oval labeled 8 dinitrogenase reductase (reduced) on the right. A red arrow pointing down from the orange oval labeled 8 dinitrogenase reductase (reduced) to an orange oval below labeled 8 dinitrogenase reductase (reduced) plus 16 A T P is joined by a gray oval showing the addition of 16 of an orange oval labeled A T P. An arrow labeled 8 minus curves down from the orange oval below labeled 8 dinitrogenase reductase (reduced) plus 16 A T P to meet a curved arrow below, at which point it becomes gray as it continues up to point to a gray oval labeled 8 dinitrogenase reductase (oxidized) plus 16 A T P. A gray arrow points up from the gray oval labeled 8 dinitrogenase reductase (oxidized) plus 16 A T P to the previously described gray oval labeled 8 dinitrogenase reductase (reduced), accompanied by an arrow showing the loss of 16 of a gray oval labeled A D P plus 16 P subscript i end subscript. The arrow pointing right from the orange oval labeled 8 dinitrogenase reductase (reduced) plus 16 A T P to 8 dinitrogenase reductase (oxidized) plus 16 A T P meets a curved arrow below from a gray oval labeled dinitrogenase (oxidized) to an orange oval labeled dinitrogenase (reduced). The two arrows meet at a point labeled 8 e minus and the bottom arrow becomes red from this point until it reaches the orange oval labeled dinitrogenase (reduced). A red arrow curves down from the orange oval labeled dinitrogenase (reduced to meet two curved arrows below before turning gray as it reaches a gray oval labeled dinitrogenase (oxidized). Both of the curved arrows below are gray on the right and red on the left. The outer curved arrow below shows the addition of N 2 in a red box and loss of 2 N H 4 plus in a red box with 6 e minus written above its red portion. The inner curved arrow shows the addition of 2 H plus and loss of H 2 with 2 e minus written above its red portion.

In the reaction carried out by dinitrogenase reductase, both ATP binding and ATP hydrolysis bring about protein conformational changes that help overcome the high activation energy of nitrogen fixation. The binding of two ATP molecules to the reductase shifts the reduction potential $(E^{'} °)$ $left-parenthesis upper E prime degree right-parenthesis$ of this protein from $- 300$ $negative 300$ to $- 420 mV$ $negative 420 mV$ , an enhancement of its reducing power that is required to transfer electrons through dinitrogenase to $N_{2}$ $upper N Subscript 2$ ; the standard reduction potential for the half-reaction $N_{2} + 6 H^{+} + 6 e^{-} → 2 {NH}_{3}$ $upper N Subscript 2 Baseline plus 6 upper H Superscript plus Baseline plus 6 e Superscript en-dash Baseline right-arrow 2 NH Subscript 3 Baseline$ is $negative 0.34 upper V$ $negative 0.34 upper V$ . The ATP molecules are then hydrolyzed just before the actual transfer of one electron to dinitrogenase.

ATP binding and hydrolysis change the conformation of nitrogenase reductase in two regions, which are structurally homologous with the switch 1 and switch 2 regions of the GTP-binding proteins involved in biological signaling (see Fig. 12-12). ATP binding produces a conformational change that brings the 4Fe-4S center of the reductase closer to the P cluster of dinitrogenase (from 18 Å to 14 Å away), which facilitates electron transfer between the reductase and dinitrogenase. The details of electron transfer from the P cluster to the FeMo cofactor, and the means by which eight electrons are accumulated by nitrogenase, are not yet known in detail. Two pathways that conform to available data, both involving the Mo atom as a central player, are illustrated in Figure 22-5.

A figure shows two possible hypotheses for intermediates involved in N 2 reduction. — FIGURE 22-5 Two reasonable hypotheses for the intermediates involved in $N_{2}$ $upper N Subscript 2$ reduction. In both scenarios, the FeMo cofactor (abbreviated as M here) plays a central role, binding directly to one of the nitrogen atoms of $N_{2}$ $upper N Subscript 2$ and remaining bound throughout the sequence of reduction steps. [Information from L. C. Seefeldt et al., *Annu. Rev. Biochem.* 78:701, 2009, Fig. 9.]

Dinitrogen is N triple bonded to N bonded to bolded M end bold, labeled F e Mo cofactor. Right- and left-pointing arrows extend to N 2, indicating a reversible reaction. An arrow points down from N triple bonded to N bonded to bolded M to diazednido, which is H N double bonded to N bonded to bolded M end bold. Arrows point to the lower left and lower right. The left-hand arrow points to H 2 N bonded to N double bonded to bolded M end bold, from which an arrow points down to nitride accompanied by a curve showing the loss of red highlighted N H 3. Nitrido is N triple bonded to bolded M end bold. An arrow points down from nitrodo to imido, which is H N double bonded to bolded M end bold. An arrow points from imido to amido at the bottom center, which is H 2 N bonded to bolded M end bold. An arrow points down from amido to amine, which is H 3 N bonded to bolded M end bold, from which an arrow points to red highlighted N H 3. The right-hand arrow from diazenido points down to H N double bonded to N H bonded to bolded M end bold, from which an arrow points down to hydrazido. Hydrazido is H 2 N bonded to N H bonded to bolded M end bold. An arrow points down from hydrazido to H 2 N bonded to N H 2 bonded to bolded M end bold, from which an arrow curves right to red highlighted N H 3 and a second arrow points down to amino at the lower left that was also produced by the left-hand series of reactions. An arrow points down from amido to amine, from which an arrow points to red highlighted N H 3.

The nitrogenase complex is remarkably unstable in the presence of oxygen. The reductase is inactivated in air, with a half-life of 30 seconds; dinitrogenase has a half-life of only 10 minutes in air. Free-living bacteria that fix nitrogen cope with this problem in a variety of ways. Some live only anaerobically or repress nitrogenase synthesis when oxygen is present. Some aerobic species, such as A. vinelandii, partially uncouple electron transfer from ATP synthesis so that oxygen is burned off as rapidly as it enters the cell (see Box 19-1). When fixing nitrogen, cultures of these bacteria increase in temperature as a result of their efforts to rid themselves of oxygen.

The symbiotic relationship between leguminous plants and the nitrogen-fixing bacteria in their root nodules (Fig. 22-6) takes care of both the energy requirements and the oxygen lability of the nitrogenase complex. The energy required for nitrogen fixation was probably the evolutionary driving force for this plant-bacteria association. The bacteria in root nodules have access to a large reservoir of energy in the form of abundant carbohydrate and citric acid cycle intermediates made available by the plant. This may allow the bacteria to fix hundreds of times more nitrogen than do their free-living cousins under conditions generally encountered in soils. To solve the oxygen-toxicity problem, the bacteria in root nodules are bathed in a solution of the oxygen-binding heme protein leghemoglobin, produced by the plant (although the heme may be contributed by the bacteria). Leghemoglobin binds all available oxygen so that it cannot interfere with nitrogen fixation, and it efficiently delivers the oxygen to the bacterial electron-transfer system. The benefit to the plant, of course, is a ready supply of reduced nitrogen. In fact, the bacterial symbionts typically produce far more ${NH}_{3}$ $NH Subscript 3$ than is needed by their symbiotic partner; the excess is released into the soil. The efficiency of the symbiosis between plants and bacteria is evident in the enrichment of soil nitrogen brought about by leguminous plants. This enrichment of ${NH}_{3}$ $NH Subscript 3$ in the soil is the basis of crop rotation methods, in which plantings of nonleguminous plants (such as maize) that extract fixed nitrogen from the soil are alternated with plantings of legumes such as alfalfa, peas, or clover.

A two-part figure shows a photo of pea plant root nodules in part a and an artificially colored electron micrograph of a thin section through a pea root nodule in part b. — FIGURE 22-6 Nitrogen-fixing nodules. (a) Pea plant (*Pisum sativum*) root nodules containing the nitrogen-fixing bacterium *Rhizobium leguminosarum*. The nodules are pink due to the presence of leghemoglobin; this heme protein has a very high binding affinity for oxygen, which strongly inhibits nitrogenase. (b) Artificially colorized electron micrograph of a thin section through a pea root nodule. Symbiotic nitrogen-fixing bacteria, or bacteroids (red), live inside the nodule cell, surrounded by the peribacteroid membrane (blue). Bacteroids produce the nitrogenase complex that converts atmospheric nitrogen ${(N}_{2})$ $left-parenthesis upper N Subscript 2 Baseline right-parenthesis$ to ammonium $({NH}_{4}^{+})$ $left-parenthesis NH Subscript 4 Superscript plus Baseline right-parenthesis$ ; without the bacteroids, the plant is unable to utilize $N_{2}$ $upper N Subscript 2$ . (The cell nucleus is shown in yellow/green. Not visible in this micrograph are other organelles of the infected root cell that are normally found in plant cells.)

FIGURE 22-6 Nitrogen-fixing nodules. (a) Pea plant (*Pisum sativum*) root nodules containing the nitrogen-fixing bacterium *Rhizobium leguminosarum*. The nodules are pink due to the presence of leghemoglobin; this heme protein has a very high binding affinity for oxygen, which strongly inhibits nitrogenase. (b) Artificially colorized electron micrograph of a thin section through a pea root nodule. Symbiotic nitrogen-fixing bacteria, or bacteroids (red), live inside the nodule cell, surrounded by the peribacteroid membrane (blue). Bacteroids produce the nitrogenase complex that converts atmospheric nitrogen ${(N}_{2})$ $left-parenthesis upper N Subscript 2 Baseline right-parenthesis$ to ammonium $({NH}_{4}^{+})$ $left-parenthesis NH Subscript 4 Superscript plus Baseline right-parenthesis$ ; without the bacteroids, the plant is unable to utilize $N_{2}$ $upper N Subscript 2$ . (The cell nucleus is shown in yellow/green. Not visible in this micrograph are other organelles of the infected root cell that are normally found in plant cells.)

Nitrogen fixation is energetically costly: 16 ATP and 8 electrons yield only 2 ${NH}_{3}$ $NH Subscript 3$ . It is therefore not surprising that the process is tightly regulated so that ${NH}_{3}$ $NH Subscript 3$ is produced only when needed. High [ADP], an indicator of low [ATP], is a strong inhibitor of nitrogenase. ${NH}_{4}^{+}$ $NH Subscript 4 Superscript plus$ represses the expression of the ~20 nitrogen fixation (nif) genes, effectively shutting down the pathway. Covalent alteration of nitrogenase is also used in some diazotrophs to control nitrogen fixation in response to the availability of ${NH}_{4}^{+}$ $NH Subscript 4 Superscript plus$ in the surroundings. Transfer of an ADP-ribosyl group from NADH to a specific Arg residue in the nitrogenase reductase shuts down $N_{2}$ $upper N Subscript 2$ fixation in Rhodospirillum, for example. This is the same covalent modification that we saw in the case of G protein inhibition by the toxins of cholera and pertussis (see Fig. 12-14).

Nitrogen fixation is the subject of intense study because of its immense practical importance. Industrial production of ammonia for use in fertilizers requires a large and expensive input of energy, and this has spurred a drive to develop recombinant or transgenic organisms that can fix nitrogen. In principle, recombinant DNA techniques (Chapter 9) might be used to transfer the DNA that encodes the enzymes of nitrogen fixation into non-nitrogen-fixing bacteria and plants. However, those genes alone will not suffice. About 20 genes are essential to nitrogenase activity in bacteria, many of them needed for the synthesis, assembly, and insertion of the cofactors. There is also the problem of protecting the enzyme in its new setting from destruction by oxygen. In all, there are formidable challenges in engineering new nitrogen-fixing plants. Success in these efforts will depend on overcoming the problem of oxygen toxicity in any cell that produces nitrogenase.

Ammonia Is Incorporated into Biomolecules through Glutamate and Glutamine

Reduced nitrogen in the form of ${NH}_{4}^{+}$ $NH Subscript 4 Superscript plus$ is assimilated into amino acids and then into other nitrogen-containing biomolecules. Two amino acids, glutamate and glutamine, provide the critical entry point. Recall that these same two amino acids play central roles in the catabolism of ammonia and amino groups in amino acid oxidation (Chapter 18). Glutamate is the source of amino groups for most other amino acids, through transamination reactions (the reverse of the reaction shown in Fig. 18-4). The amide nitrogen of glutamine is a source of amino groups in a wide range of biosynthetic processes. In most types of cells, and in extracellular fluids in higher organisms, one or both of these amino acids are present at higher concentrations — sometimes an order of magnitude or more higher — than other amino acids. An Escherichia coli cell requires so much glutamate that this amino acid is one of the primary solutes in the cytosol. Its concentration is regulated not only in response to the cell’s nitrogen requirements but also to maintain an osmotic balance between the cytosol and the external medium.

The biosynthetic pathways to glutamate and glutamine are simple, and all or some of the steps occur in most organisms. The most important pathway for the assimilation of ${NH}_{4}^{+}$ $NH Subscript 4 Superscript plus$ into glutamate requires two reactions. The net effect is to convert glutamate, α-ketoglutarate, and ammonia into two molecules of glutamate.

First, ${NH}_{4}^{+}$ $NH Subscript 4 Superscript plus$ is reacted with glutamate to produce glutamine, using the enzyme glutamine synthetase. This reaction takes place in two steps, with enzyme-bound γ-glutamyl phosphate as an intermediate (see Fig. 18-8):

\frac{\begin{array}{l} (1) Glutamate + ATP \to γ -glutamyl phosphate + ADP \\ (2) γ -Glutamyl phosphate + {NH}_{4}^{+} \to glutamine + P_{i} + H^{+} \end{array}}{\begin{array}{l} S u m : Glutamate + {NH}_{4}^{+} + ATP \to glutamine + ADP + P_{i} + H^{+} \end{array}}

$StartFraction StartLayout 1st Row left-parenthesis 1 right-parenthesis Glutamate plus ATP right-arrow gamma hyphen glutamyl phosphate plus ADP 2nd Row left-parenthesis 2 right-parenthesis gamma hyphen Glutamyl phosphate plus NH Subscript 4 Superscript plus Baseline right-arrow glutamine plus upper P Subscript i Baseline plus upper H Superscript plus Baseline EndLayout Over StartLayout 1st Row upper S u m colon Glutamate plus NH Subscript 4 Superscript plus Baseline plus ATP right-arrow glutamine plus ADP plus upper P Subscript i Baseline plus upper H Superscript plus Baseline EndLayout EndFraction$

(22-1)

Glutamine synthetase is found in all organisms. In addition to its importance for ${NH}_{4}^{+}$ $NH Subscript 4 Superscript plus$ assimilation in bacteria, it has a central role in amino acid metabolism in mammals, converting free ${NH}_{4}^{+}$ $NH Subscript 4 Superscript plus$ , which is toxic, to glutamine for transport in the blood (Chapter 18).

In the second reaction needed for ${NH}_{4}^{+}$ $NH Subscript 4 Superscript plus$ assimilation, the glutamine reacts with α-ketoglutarate to generate two molecules of glutamate. In bacteria and plants, this reaction is catalyzed by glutamate synthase. (An alternative name for this enzyme, glutamate:oxoglutarate aminotransferase, yields the acronym GOGAT, by which the enzyme also is known.) α-Ketoglutarate, an intermediate of the citric acid cycle, undergoes reductive amination with glutamine as nitrogen donor:

α -Ketoglutarate + glutamine + NAD (P) H + H^{+} \to 2 glutamate + {NAD(P)}^{+}

$alpha hyphen Ketoglutarate plus glutamine plus NAD left-parenthesis upper P right-parenthesis upper H plus upper H Superscript plus Baseline right-arrow 2 glutamate plus NAD left-parenthesis upper P right-parenthesis Superscript plus$

(22-2)

The net reaction of glutamine synthetase and glutamate synthase (Eqns 22-1 and 22-2) is

α -Ketoglutarate + {NH}_{4}^{+} + NAD(P)H + ATP \to glutamate + {NAD(P)}^{+} + ADP + P_{i}

$alpha hyphen Ketoglutarate plus NH Subscript 4 Superscript plus Baseline plus NAD left-parenthesis upper P right-parenthesis upper H plus ATP right-arrow glutamate plus NAD left-parenthesis upper P right-parenthesis Superscript plus Baseline plus ADP plus upper P Subscript i Baseline$

Glutamate synthase is not present in animals, which instead maintain high levels of glutamate by processes such as the transamination of α-ketoglutarate during amino acid catabolism. Plants possess a second alternative form of glutamate synthase that uses reduced ferredoxin rather than NADPH as a source of reducing electrons.

Glutamate can also be formed in yet another, albeit minor, pathway: the reaction of α-ketoglutarate and ${NH}_{4}^{+}$ $NH Subscript 4 Superscript plus$ to form glutamate in one step. This is catalyzed by glutamate dehydrogenase, an enzyme present in all organisms. Reducing power is furnished by NADPH:

α -Ketoglutarate + {NH}_{4}^{+} + NAD (P) H + H^{+} \to glutamate + {NAD(P)}^{+} + H_{2} O

$alpha hyphen Ketoglutarate plus NH Subscript 4 Superscript plus Baseline plus NAD left-parenthesis upper P right-parenthesis upper H plus upper H Superscript plus Baseline right-arrow glutamate plus NAD left-parenthesis upper P right-parenthesis Superscript plus Baseline plus upper H Subscript 2 Baseline upper O$

We encountered this reaction in the catabolism of amino acids (see Fig. 18-7). In eukaryotic cells, glutamate dehydrogenase is located in the mitochondrial matrix. The reaction equilibrium favors the reactants, and the $K_{m}$ $upper K Subscript m$ for ${NH}_{4}^{+}$ $NH Subscript 4 Superscript plus$ $(\sim 1 mM)$ $left-parenthesis tilde 1 mM right-parenthesis$ is so high that the reaction is not important for ${NH}_{4}^{+}$ $NH Subscript 4 Superscript plus$ assimilation in mammals. (Recall that the glutamate dehydrogenase reaction, in reverse (see Fig. 18-10), is one source of ${NH}_{4}^{+}$ $NH Subscript 4 Superscript plus$ destined for the urea cycle.) In microorganisms and plants, concentrations of ${NH}_{4}^{+}$ $NH Subscript 4 Superscript plus$ high enough for the glutamate dehydrogenase reaction to make a significant contribution to glutamate levels generally occur only when ${NH}_{3}$ $NH Subscript 3$ is added artificially to the growth environment. In general, soil bacteria and plants rely on the two-enzyme pathway outlined above (Eqns 22-1, 22-2).

Glutamine Synthetase Is a Primary Regulatory Point in Nitrogen Metabolism

There are three known classes of glutamine synthetases. The class I enzyme (GSI, found in bacteria) has 12 identical subunits of $M_{r} 50, 000$ $upper M Subscript r Baseline 50 comma 000$ (Fig. 22-7) and is regulated both allosterically and by covalent modification. The class II enzyme (GSII, found in eukaryotes and some bacteria) has 10 identical subunits. The third class of glutamine synthetases (GSIII), so far found only in two bacterial species, are much larger enzymes, consisting of a double-ringed dodecamer of identical chains.

The subunit structure of bacterial type Roman numeral 1 glutamine synthetase is shown. — FIGURE 22-7 Subunit structure of bacterial type I glutamine synthetase. This view shows 6 of the 12 identical subunits; a second layer of 6 subunits lies directly beneath those shown. Each of the 12 subunits has an active site, where ATP and glutamate are bound in orientations that favor transfer of a phosphoryl group from ATP to the side-chain carboxyl of glutamate. In this crystal structure, ADP occupies the ATP site. [Data from PDB ID 2GLS, M. M. Yamashita et al., *J. Biol. Chem.* 264:17,681, 1989.]

Befitting their central metabolic role as an entry point for reduced nitrogen, GSI glutamine synthetases are highly regulated. In enteric bacteria such as E. coli, the regulation is unusually complex. Alanine, glycine, and at least six end products of glutamine metabolism are allosteric inhibitors (Fig. 22-8). Each inhibitor alone produces only partial inhibition, but the effects of multiple inhibitors are more than additive, and all eight together virtually shut down the enzyme. This is an example of cumulative feedback inhibition. This control mechanism provides a constant adjustment of glutamine levels to match immediate metabolic requirements.

A figure shows allosteric regulation of glutamine synthesis from glutamate. — FIGURE 22-8 Allosteric regulation of glutamine synthetase. The enzyme undergoes cumulative regulation by six end products of glutamine metabolism. Alanine and glycine probably serve as indicators of the general status of amino acid metabolism in the cell.

A yellow box labeled glutamate is shown at the top center. A long vertical arrow labeled glutamine synthesis extends down to a yellow box labeled glutamine near the bottom of the figure. Just below glutamate, a curved line shows the addition of N H 3. Just below the addition of N H 3, a curved arrow shows the addition of an orange oval labeled A T P and loss of A D P plus P subscript i end subscript. Arrows point from glutamine to A T P, tryptophan, carbamoyl phosphate, C T P, histidine, and glucosamine 6-phosphate. From top to bottom along the arrow pointing from glutamate to glutamine, regulation occurs as follows. A red “X” in a red circle just above the center is indicated by a dashed arrow from glycine. A red “X” in a red circle just below is indicated by a dashed arrow from alanine. Just below, a red “X” in a red circle on the left is indicated by a dashed arrow from carbamoyl phosphate and a similar red “X” in a red circle on the right is indicated by glucosamine 6-phosphate. Just below, a red “X” in a red circle on the left is indicated by a dashed arrow from tryptophan and a similar red “X” in a red circle on the right is indicated by histidine. Just below, near the bottom of the arrow, a red “X” in a red circle on the left is indicated by a dashed arrow from A M P and a similar red “X” in a red circle on the right is indicated by C T P.

Superimposed on the allosteric regulation is inhibition by adenylylation of (addition of AMP to) ${Tyr}^{397}$ $Tyr Superscript 397$ , located near the enzyme’s active site (Fig. 22-9). This covalent modification increases sensitivity to the allosteric inhibitors, and activity decreases as more subunits are adenylylated. Both adenylylation and de-adenylylation are promoted by adenylyltransferase (AT in Fig. 22-9), part of a complex enzymatic cascade that responds to levels of glutamine, α-ketoglutarate, ATP, and $P_{i}$ $upper P Subscript i$ . The activity of adenylyltransferase is modulated by binding to a regulatory protein called $P_{II}$ $upper P Subscript II$ , and the activity of $P_{II}$ $upper P Subscript II$ , in turn, is regulated by covalent modification (uridylylation), again at a Tyr residue. The adenylyltransferase complex with uridylylated $P_{II}$ $upper P Subscript II$ ( $P_{II}$ $upper P Subscript II$ -UMP) stimulates de-adenylylation, whereas the same complex with deuridylylated $P_{II}$ $upper P Subscript II$ stimulates adenylylation of glutamine synthetase. Both uridylylation and deuridylylation of $P_{II}$ $upper P Subscript II$ are brought about by a single enzyme, uridylyltransferase. Uridylylation is inhibited by binding of glutamine and $P_{i}$ $upper P Subscript i$ to uridylyltransferase and is stimulated by binding of α-ketoglutarate and ATP to $P_{II}$ $upper P Subscript II$ .

A figure illustrates second level regulation of glutamine synthetase by showing an adenylated T y r residue in part a and the cascade leading to the adenylylation of glutamine synthetase, resulting in inactivation, in part b. — FIGURE 22-9 Second level of regulation of glutamine synthetase: covalent modifications. (a) An adenylylated Tyr residue. (b) Cascade leading to adenylylation (inactivation) of glutamine synthetase. AT represents adenylyltransferase; UT, uridylyltransferase. $P_{II}$ $upper P Subscript II$ is a regulatory protein, itself regulated by uridylylation.

Part a shows a blue piece of enzyme on the left with a vertical boundary near the middle of the illustration that has an invagination in its center that contains O further bound to phosphate to the right and to tyrosine to the left. A benzene ring labeled T y r in the blue region has a left side vertex bonded to C H 2 bonded to enzyme and a right vertex bonded to O outside of the blue region that is further bonded to P double bonded to O above, bonded to O minus below, and bonded to O to the right further bonded to C 5 prime bonded to 2 H and to C 4 prime of a five-membered ring below. The ring has O at its top vertex, C 1 prime bonded to a box labeled adenine above and H below; C 2 prime and C 3 prime bonded to H above and O H below; C 4 prime bonded to H below and C 5 prime above bonded to 2 H and to O leading to phosphate to its left. Part b shows a yellow box labeled alpha-ketoglutarate near the top with a short arrow pointing down to a yellow box labeled glutamate, from which a longer arrow points down to a yellow box labeled glutamine and is accompanied by a curved line showing the addition of N H 2 and a curved arrow showing the addition of A T P and loss of A D P plus P subscript i end subscript. P subscript Roman numeral 2 end subscript to the lower right is a purple sphere further bonded to U M P to the lower left with arrows pointing in two directions. Upward- and downward-pointing arrows extend up to a similar purple sphere labeled P subscript Roman numeral 2 end subscript that is not bonded to U M P, indicating a reversible reaction. A curved arrow meets the upward arrow to show the addition of H 2 O and loss of U M P. A second curved arrow meets the downward arrow to show the addition of U T P and loss of P P subscript i end subscript and a blue circle labeled U T above the word uridylylation is at the inflection point of this arrow. Dashed arrows point from alpha-ketoglutarate and A T P from the reactions converting alpha-ketoglutarate to glutamine to a green triangle enclosed in a green circle near the top of the curved arrow that adds U T P to participate in the downward reaction. A dashed arrow from glutamine indicates a red “X” in a red circle near the bottom of the curved arrow from U T P to P P subscript i end subscript. An arrow extends left from the purple sphere labeled P Roman numeral subscript 2 end subscript and is joined by a blue oval labeled A T before reaching a complex of the purple sphere labeled P Roman numeral subscript 2 end subscript and the blue oval labeled A T, which is at the upper right of the sphere. An arrow points down from this complex to the place where a curved arrow labeled adenylylation from glutamine synthetase (active) to glutamine synthetase inactive meets a curved arrow showing the addition of A T P and loss of P P subscript i end subscript. Glutamine synthetase (active) is shown as a blue oval with short lines radiating from all sides and glutamine synthetase (inactive) is shown as a similar oval with no radiating lines and a short bond to A M P at the lower left. An arrow labeled deadenylylation curves from glutamine synthetase (inactive) to glutamine synthetase (active) and is accompanied by a curved arrow showing the addition of P subscript i end subscript and loss of A D P. An arrow points up to the inflection point of this curve from a complex of A T and P subscript Roman numeral 2 end subscript bonded to U M. This complex is indicated by an arrow from the purple sphere labeled P subscript Roman numeral 2 end subscript bonded to U M P at its lower left and joined by a curved line indicating the addition of A T.

The regulation does not stop there. The uridylylated $P_{II}$ $upper P Subscript II$ also mediates the activation of transcription of the gene encoding glutamine synthetase, thus increasing the cellular concentration of the enzyme; the deuridylylated $P_{II}$ $upper P Subscript II$ brings about a decrease in transcription of the same gene. This mechanism involves an interaction of $P_{II}$ $upper P Subscript II$ with additional proteins involved in gene regulation, of a type described in Chapter 28. The net result of this elaborate system of controls is a decrease in glutamine synthetase activity when glutamine levels are high, and an increase in activity when glutamine levels are low and α-ketoglutarate and ATP (substrates for the synthetase reaction) are available. The multiple layers of regulation permit a sensitive response in which glutamine synthesis is tailored to cellular needs.

Several Classes of Reactions Play Special Roles in the Biosynthesis of Amino Acids and Nucleotides

The pathways described in this chapter include a variety of interesting chemical rearrangements. Several of these recur and deserve special note before we progress to the pathways themselves. These are (1) transamination reactions and other rearrangements promoted by enzymes containing pyridoxal phosphate; (2) transfer of one-carbon groups, with either tetrahydrofolate (usually at the $— CHO$ $em-dash CHO$ and $— {CH}_{2} OH$ $em-dash CH Subscript 2 Baseline OH$ oxidation levels) or S-adenosylmethionine (at the $— {CH}_{3}$ $em-dash CH Subscript 3 Baseline$ oxidation level) as cofactor; and (3) transfer of amino groups derived from the amide nitrogen of glutamine. Pyridoxal phosphate (PLP), tetrahydrofolate ( $H_{4}$ $upper H Subscript 4$ folate), and S-adenosylmethionine (adoMet) are described in some detail in Chapter 18 (see Figs. 18-6, 18-17, and 18-18). Here we focus on amino group transfer involving the amide nitrogen of glutamine.

More than a dozen known biosynthetic reactions use glutamine as the major physiological source of amino groups, and most of these occur in the pathways outlined in this chapter. As a class, the enzymes catalyzing these reactions are called glutamine amidotransferases. All have two structural domains: one binding glutamine, the other binding the second substrate, which serves as amino group acceptor (Fig. 22-10). A conserved Cys residue in the glutamine-binding domain is believed to act as a nucleophile, cleaving the amide bond of glutamine and forming a covalent glutamyl-enzyme intermediate. The ${NH}_{3}$ $NH Subscript 3$ produced in this reaction is not released, but instead is transferred through an “ammonia channel” to a second active site, where it reacts with the second substrate to form the aminated product. The covalent intermediate is hydrolyzed to the free enzyme and glutamate. If the second substrate must be activated, the usual method is the use of ATP to generate an acyl phosphate intermediate ( $R — OX$ $upper R em-dash OX$ in Fig. 22-10, where X is a phosphoryl group). The enzyme glutaminase acts similarly but uses $H_{2} O$ $upper H Subscript 2 Baseline upper O$ as the second substrate, yielding ${NH}_{4}^{+}$ $NH Subscript 4 Superscript plus$ and glutamate (see Fig. 18-8).

A figure shows a proposed mechanism for glutamine amidotransferases. — MECHANISM FIGURE 22-10 Proposed mechanism for glutamine amidotransferases. Each enzyme has two domains. The glutamine-binding domain contains structural elements conserved among many of these enzymes, including a Cys residue required for activity. The ${NH}_{3}$ $NH Subscript 3$ -acceptor (second-substrate) domain varies. Two types of amino acceptors are shown. X represents an activating group, typically a phosphoryl group derived from ATP, that facilitates displacement of a hydroxyl group from $R — OH$ $upper R em-dash OH$ by ${NH}_{3}$ $NH Subscript 3$ .

MECHANISM FIGURE 22-10 Proposed mechanism for glutamine amidotransferases. Each enzyme has two domains. The glutamine-binding domain contains structural elements conserved among many of these enzymes, including a Cys residue required for activity. The ${NH}_{3}$ $NH Subscript 3$ -acceptor (second-substrate) domain varies. Two types of amino acceptors are shown. X represents an activating group, typically a phosphoryl group derived from ATP, that facilitates displacement of a hydroxyl group from $R — OH$ $upper R em-dash OH$ by ${NH}_{3}$ $NH Subscript 3$ .

A blue structure labeled glutamine amidotransferase has two circular openings with protrusions from the top and bottom center that do not form a full central wall. The opening between these protrusions is labeled N H 3 channel. The left-hand side is labeled glutamine-binding domain and the right-hand side is labeled N H 3-acceptor domain. C y s in the lower right side of the left-hand ring has a bond to S H in the opening with a red pair of electrons on S. Glutamine is shown as a vertical chain. Numbered from top to bottom, it has C 1 forming C O O minus, C 2 bonded to H and to N H 3 with a positive charge on N; C 3 and C 4 bonded to 2 H; and C 5 double bonded to O to the lower left and to red highlighted N H 2 to the lower right. Red arrows point from the red pair of electrons on S to C 5 and from the bond between C 5 and red highlighted N H 2 to H plus. Step 1: The gamma-amido nitrogen of glutamine (red) is released as N H 3 in a reaction that probably involves a covalent glutamyl-enzyme intermediate. The N H 3 travels via a channel to the second active site. An arrow points down accompanied by a curved line showing the addition of an acceptor, R bonded to O H or C bonded to R superscript 1 end superscript to the upper left, R superscript 2 end superscript to the lower left, and double bonded to O to the right. A similar structure with two chambers is labeled, glutamyl-enzyme intermediate. C y s at the lower right is still bonded to S in the open area, but S is now bonded to C 5. C 5 is also double bonded to O but no longer bonded to N H 2. A dashed line shows the movement of red highlighted N H 3 into the right opening of the structure. The opening of the right side of the structure contains activated substrate, R bonded to O X or C bonded to R superscript 1 end superscript to the upper left, R superscript 2 end superscript to the lower left, and double bonded to O to the right. Step 2: N H 3 reacts with any of several acceptors. An arrow points down to two sets of products and is accompanied by a curved arrow showing the addition of H 2 O and loss of glutamate. The first set of products is R bonded to red highlighted N H 2 plus H bonded to O X. The word “or” separates these from the second set of products, which includes C bonded to R superscript 1 end superscript to the upper left, R superscript 2 end superscript to the lower left, and double bonded to red highlighted N H to the right plus H 2 O.

SUMMARY 22.1 Overview of Nitrogen Metabolism

The molecular nitrogen that makes up 80% of Earth’s atmosphere is unavailable to most living organisms until it is reduced. A complex web of reactions converts atmospheric $N_{2}$ $upper N Subscript 2$ to biologically useful forms and maintains a global balance between them. Prominent species that are interconverted within this web include ammonia ( ${NH}_{3}$ $NH Subscript 3$ or ${NH}_{4}^{+}$ $NH Subscript 4 Superscript plus$ ; most reduced), nitrite $({NO}_{2}^{-})$ $left-parenthesis NO Subscript 2 Superscript minus Baseline right-parenthesis$ , and nitrate ( ${NO}_{3}^{-}$ $NO Subscript 3 Superscript minus$ ; most oxidized). Conversion of $N_{2}$ $upper N Subscript 2$ to ammonia is fixation. Nitrification constitutes the steps converting ammonia to nitrate. Conversion of nitrate to $N_{2}$ $upper N Subscript 2$ constitutes denitrification. The alternative conversion of nitrate to ammonia is nitrate assimilation.
Fixation of $N_{2}$ $upper N Subscript 2$ as ${NH}_{3}$ $NH Subscript 3$ is carried out by the nitrogenase complex, in a reaction that requires large investments of ATP and of reducing power. The nitrogenase complex is highly labile in the presence of $O_{2}$ $upper O Subscript 2$ , and it is subject to regulation by the supply of ${NH}_{3}$ $NH Subscript 3$ .
In living systems, reduced nitrogen is incorporated first into amino acids and then into a variety of other biomolecules, including nucleotides. The key entry point is the amino acid glutamate. Glutamate and glutamine are the nitrogen donors in a wide range of biosynthetic reactions.
Glutamine synthetase, which catalyzes the formation of glutamine from glutamate, is a main regulatory enzyme of nitrogen metabolism.
The amino acid and nucleotide biosynthetic pathways make repeated use of the biological cofactors pyridoxal phosphate, tetrahydrofolate, and S-adenosylmethionine. Pyridoxal phosphate is required for transamination reactions involving glutamate and for other amino acid transformations. One-carbon transfers require S-adenosylmethionine and tetrahydrofolate. Glutamine amidotransferases catalyze reactions that incorporate nitrogen derived from the amide group of glutamine.