18.1: The Biochemistry of Nitrogen in the Biosphere

Introduction

Organic chemistry is usually described as the chemistry of carbon-containing molecules. But isn't that definition a bit carbon-centric, especially since the prevalence of oxygen-containing molecules is staggering? What about nitrogen? We live in a dinitrogen-rich atmosphere (80%), and all classes of biomolecules (lipids, carbohydrates, nucleic acids, and proteins) contain nitrogen. Dinitrogen is very stable due to its triple bond and nonpolar nature. We rely on a few organisms to fix N₂ from the atmosphere to form ammonium (NH₄⁺), which, through nitrification and denitrification, can form nitrite (NO₂^-), nitrate (NO₃^-), nitric oxide (NO), and nitrous oxide (N₂O), the latter being a potent greenhouse gas. In the next section, we'll concentrate on the metabolic fate of amino groups in amino acids and proteins. Before exploring their fates, examine the figure below, which provides an overview of the biological nitrogen cycle. The study of biochemistry should encompass more than homo sapiens and expand to the ecosystem in which we are such a small but damaging part.

Let's break down the diagram from a biochemical perspective. Aerobic and anaerobic processes (conducted by bacteria) occur. Nitrogen-containing substances include inorganic (ammonium, nitrate, nitrite) and organic (amino acids, nucleotides, etc.) molecules. The reactions shown are both oxidative and reductive (note: the oxidation numbers of the nitrogen atoms in the molecules are shown in red). Most reactions occur underground, carried out by bacterial and Archaeal microorganisms.

Here are some of the major reactions:

• N₂ fixation (a reduction): N₂ from the air is converted by bacteria to ammonium (NH₄⁺) by the enzyme nitrogenase of soil prokaryotes. The energetically unfavorable reaction requires a lot of ATP.  Primary producers, such as plants, can then take up the ammonium and incorporate it into biomolecules such as amino acids, which animals consume. Consider this for those who may still believe that people have marginal effects on our biosphere. We may soon fix more N₂ (to NH₃) through the industrial Haber-Bosch reaction (used for fertilizer and explosive production) than all the N₂ fixed by the biosphere. A significant portion of the nitrogen used comes from the Haber-Bosch reaction. The excess NH₄⁺ (up to 50%) produced industrially, which enters the soil through fertilizers (mostly as NH₄NO₃), has overwhelmed nature's ability to balance the nitrogen cycle and is not taken up by plants. Microorganisms metabolize it into nitrite and nitrate.

• Nitrification: Ammonium is converted to nitrite by ammonia-oxidizing aerobic microorganisms and further to nitrate by a separate group of nitrite-oxidizing aerobic bacteria. Here are the reactions (Rx 1 and 2) to produce nitrate through a hydroxylamine intermediate, followed by nitrate formation (Rx 3).

\[\ce{NH3 + O2 + 2e^{-} -> NH2OH + H2O} \tag{Rx 1} \]

\[\ce{NH2OH + H2O -> NO2^{-} + 5H^{+} + 4e^{-}} \tag{Rx 2} \]

\[\ce{NO2^{-} + 1/2 O2 -> NO3^{-}} \tag{Rx 3} \]

These added ions exceed soil capacity and end up in runoff water, polluting our rivers and lakes.

• Denitrification: This anaerobic reaction pathway produces N₂ from nitrate. Here is the net reaction:

\[\ce{2NO3^{-} + 10e^{-} + 12H^{+} -> 2N2 + 6H2O} \nonumber \]

• Anammox reaction: This recently discovered bacterial anaerobic reaction pathway converts ammonium and nitrate to N₂. Here is the net reaction

\[\ce{NO2^{-} + NH3^{+} -> N2 + 2H2O} \nonumber \]

• Ammonification: This occurs when plants and animals decompose, which returns ammonium to the soil for reuse by plants and microbes.

These reactions are shown in the abbreviated Nitrogen Cycle below.

Nitrogen metabolites are essential nutrients for plants and perhaps the most important in regulating plant growth (primary productivity) and biodiversity in the biosphere. All living organisms require feedstocks to produce energy and as substrates for biosynthetic reactions. The types used depend on the organism. Plants are primary producers, so they use the carbohydrates they synthesize for energy and biosynthesis. For carnivores, proteins and their derived amino acids provide energy (through oxidation) and serve as biosynthetic precursors. For omnivorous organisms, the energy source depends on the "fed" state. With abundant food resources, carbohydrates and lipids serve as the primary energy sources. Unlike carbohydrates and lipids, which can be stored as glycogen and triacylglycerols for future use, excess protein and its associated amino acids can not be stored, so amino acids can be eliminated or used for oxidative energy.

In the fed state, carbohydrates are the main energy source, while lipids take a predominant role in the unfed state. Under starvation conditions, the organism breaks down its own proteins and uses them for oxidative energy production and residual biosynthesis. In diseased states like diabetes, which can be likened to starvation despite abundant carbohydrates, both lipids and amino acids become energy sources.

How are amino acids in animals oxidatively metabolized? Several pathways can achieve this. Still, it would seem logical that NH₄⁺ would be removed, and the carbons in the remaining molecule would eventually enter glycolysis or the TCA cycle in the form of ketoacids. NH₄⁺ is toxic in high concentrations. In humans, ammonium is not oxidized to nitrite or nitrate, unlike in soil, where microorganisms oxidize it. It can be recycled into nucleotides or amino acids, removing excess from the organism. Both processes must be highly controlled. We will turn our attention to the oxidation of amino acids in the next section.

Summary

(Summary written by Claude, Sonnet 4.6, Anthropic)

This chapter provides the biogeochemical and metabolic context for nitrogen metabolism, tracing the global biological nitrogen cycle and establishing the framework for understanding amino acid catabolism in animals.

Nitrogen is an essential element in all major biomolecule classes — amino acids, nucleotides, lipids, and carbohydrates all contain nitrogen — yet the most abundant atmospheric form, dinitrogen (N₂), is extraordinarily chemically inert. The N≡N triple bond (bond dissociation energy ~945 kJ/mol) and the nonpolar nature of N₂ create a prohibitively large activation energy barrier that prevents spontaneous reaction with biological or industrial reagents under mild conditions. This kinetic inertness — analogous to the kinetic sluggishness of O₂ despite its thermodynamic reactivity — means that the nitrogen cycle is entirely dependent on specialized enzymatic and industrial processes to convert N₂ into biologically accessible forms.

The biological nitrogen cycle encompasses five major transformations. Nitrogen fixation, carried out by the enzyme nitrogenase in soil prokaryotes and cyanobacteria, converts N₂ to NH₄⁺ via an energetically expensive reductive process that requires substantial ATP. Fixed ammonium is assimilated by primary producers into amino acids and nucleotides, which are then consumed by animals. Nitrification converts NH₄⁺ to NO₂⁻ and then NO₃⁻ through sequential aerobic oxidations by two specialized groups of bacteria, with hydroxylamine as an intermediate. Denitrification returns nitrogen to the atmosphere as N₂ through the anaerobic reduction of NO₃⁻. The recently discovered anammox pathway also produces N₂ by combining NH₄⁺ and NO₂⁻ anaerobically. Finally, ammonification returns NH₄⁺ to the soil through decomposition of organic matter, completing the cycle.

The industrial Haber-Bosch process mirrors biological nitrogen fixation in outcome but differs dramatically in mechanism and scale. The reaction N₂ + 3H₂ → 2NH₃ is thermodynamically favorable at room temperature (ΔG° = −16.4 kJ/mol, ΔH° = −46.2 kJ/mol) but kinetically blocked by the high activation energy required to break the N≡N bond. Because the reaction is exothermic, raising the temperature drives the equilibrium toward the reactants; however, high temperatures are necessary to achieve kinetically practical reaction rates. This conflict is resolved by using high pressure (which shifts the equilibrium toward the two-molecule product side, consistent with Le Châtelier's principle, since four moles of gas produce two), and a complex magnetite (Fe₃O₄) catalyst doped with metal oxides to prevent over-reduction of iron, which together enable economically viable ammonia production. The Haber-Bosch process now fixes enough nitrogen to support approximately half of the world's population through fertilizer production, likely matching or exceeding total biological fixation. However, less than half of this fixed nitrogen is taken up by crops; the excess enters soil and water as NH₄⁺, NO₂⁻, and NO₃⁻, causing aquatic eutrophication and contributing to the production of N₂O — a greenhouse gas far more potent than CO₂ — through microbial denitrification. Human perturbation of the nitrogen cycle, therefore, constitutes one of the most significant planetary boundary transgressions of the current era.

The chapter concludes by situating amino acid catabolism within the broader context of organismal energy metabolism. Unlike glycogen (the storage form of carbohydrate) and triacylglycerols (the storage form of lipid), there is no dedicated storage pool of amino acids in animals. Animals cannot retain excess dietary protein and its amino acids, so they must be catabolized or excreted. The metabolic state of the organism determines the extent of amino acid catabolism: in the fed state, carbohydrates are the primary energy source; in the fasted or starving state, lipids predominate, and amino acids from protein breakdown contribute significantly; in pathological states such as insulin-deficient diabetes — metabolically similar to starvation despite glucose abundance — amino acids become important energy substrates alongside lipids. The general strategy for amino acid catabolism in animals involves two conceptually distinct phases: (1) removal of the amino group, generating toxic NH₄⁺ that must be rapidly detoxified and eliminated (through pathways to be discussed in subsequent sections), and (2) oxidative metabolism of the remaining carbon skeleton, which enters either glycolysis or the TCA cycle as an appropriate ketoacid intermediate depending on the amino acid. Because NH₄⁺ is toxic at elevated concentrations and is not oxidized to nitrite or nitrate in mammals (unlike in soil bacteria), it must be metabolically converted to a non-toxic, excretable form — a process that requires tight regulation and will be explored in detail in subsequent sections of this chapter.