18.1: The Biochemistry of Nitrogen in the Biosphere

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Learning Goals (ChatGPT o3-mini)

1. Understand the Broader Scope of Organic Chemistry:

   • Explain why the traditional definition of organic chemistry as the study of carbon-containing molecules is limited, and describe the importance of nitrogen and oxygen in the structure and function of biomolecules.

2. Describe the Biological Nitrogen Cycle:

   • Outline the key steps of the nitrogen cycle, including nitrogen fixation, nitrification, denitrification, anammox, and ammonification.
   • Identify the roles of both inorganic nitrogen species (ammonium, nitrite, nitrate) and organic nitrogen compounds (amino acids, nucleotides) in the cycle.

3. Compare Biological and Industrial Nitrogen Fixation:

   • Explain how atmospheric dinitrogen (N₂) is converted to ammonium (NH₄⁺) by nitrogenase in certain microorganisms, and contrast this with the industrial Haber-Bosch process, including the thermodynamic and kinetic challenges involved.

4. Analyze Thermodynamic Considerations in Nitrogen Fixation:

   • Discuss the thermodynamic parameters (ΔH°, ΔS°, ΔG°) associated with the conversion of N₂ and H₂ to ammonia and explain why, despite being thermodynamically favorable at room temperature, the reaction is kinetically slow.

5. Evaluate the Ecological and Biospheric Impact of the Nitrogen Cycle:

   • Analyze the importance of nitrogen-containing metabolites as essential nutrients for plants, and discuss how these compounds regulate primary productivity and biodiversity in ecosystems.

6. Integrate Nitrogen Metabolism with Energy and Biosynthesis:

   • Compare how different organisms (e.g., plants as primary producers, carnivores, and omnivores) use nitrogen-containing biomolecules for energy production and biosynthetic pathways.
   • Explain why excess protein cannot be stored like carbohydrates or lipids, and how amino acid catabolism must be tightly regulated to manage toxic ammonium levels.

7. Link Amino Acid Oxidation to Metabolic Regulation:

   • Discuss the oxidative metabolism of amino acids in animals, including the removal of the amino group (forming NH₄⁺) and the subsequent conversion of the carbon skeleton into ketoacids that feed into glycolysis or the TCA cycle.
   • Understand the mechanisms by which ammonium is recycled or eliminated, emphasizing its toxicity and the need for precise regulation.

These learning goals will equip students with a comprehensive understanding of the nitrogen cycle’s key reactions, its interplay with broader metabolic processes, and its ecological significance.

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, given its triple bond and its 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, look at the figure below, which shows an overall view 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. There are aerobic and anaerobic processes (conducted by bacteria). Nitrogen-containing substances include inorganic (ammonium, nitrate, nitrite) and organic (amino acids, nucleotides, etc) molecules. The reactions shown are oxidative and reductive (note: the oxidation number of the nitrogen atoms in the molecules is shown in red). Most of the reactions are carried out underground 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 disfavored reaction requires lots of ATP.  Ammonium once made can then be taken up by primary producers like plants and incorporated 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 Born-Haber reaction (used for fertilizer and explosive productions) that all the N₂ fixed by the biosphere. Much of the nitrogen in use comes from the Haber-Bosch reaction. The excess NH₄⁺ (upwards of 50%) produced industrially and which enters the soil in fertilizers (mostly as NH₄NO₃) has overwhelmed nature's ability to balance the nitrogen cycle and is not taken up by plants. It is metabolized by microorganisms to nitrite and nitrate.

Haber-Bosch fixation of N₂

Dinitrogen, N₂, has a triple bond, making N₂ extraordinarily stable, thus explaining why we can breathe an atmosphere containing 80% N₂ and not die. You may remember from introductory chemistry that high pressure and temperature are needed in the Haber-Bosch process to convert N₂ and H₂ to ammonia, NH₃. As with any scientific advance, the Haber-Bosch process has brought harm (it's used for explosive weapons) and good (fertilizers). This process now fixes enough N₂ in the form of fertilizers to support half of the world’s population, with nitrogenase supporting the rest. Efforts are underway to modify plants to make nitrogenase genetically, eliminating the need for fertilizers but perhaps creating unforeseen problems.

You might be surprised that the equilibrium constant favors ammonia formation at room temperature; hence, ΔG⁰ < 0. The reaction is favored enthalpically as it is exothermic at room temperature. It is disfavored entropically, as should be evident from the balanced equation:

\[\ce{N2(g) + 3H2(g) → 2 NH3(g)}. \nonumber \]

The thermodynamic parameters for the reaction (per mol) are ΔH° = –46.2 kJ, ΔS° = –389 J K–1, and ΔG° = –16.4 kJ at 298 K

The entropy is negative since the reaction proceeds from four molecules to two molecules. From an enthalpy perspective, raising the temperature of an exothermic reaction reverses it. Increasing the pressure shifts the equilibrium to the side with the fewest molecules.

If the reaction is favored thermodynamically at room temperature, why doesn’t it proceed readily? This story sounds familiar, as this same descriptor applies to the oxidation of organic molecules with dioxygen. There, we showed using MO theory that the reaction is kinetically slow. The same is true for NH₃ formation. A superficial way to see this is that we must break bonds in the stable N₂ to start the reaction, leading to a high activation energy and making the reaction kinetics sluggish.

One could jump-start the reaction by raising the temperature, slowing an exothermic reaction. The Keq and ΔG⁰ are functions of temperature, and for this reaction, the reaction becomes disfavored at higher temperatures. The solution Haber found was high pressure, forcing the reaction to the side with fewer gas molecules, and high temperature to overcome the activation energy barrier and make the reaction kinetically feasible. A complex metal catalyst (magnetite - Fe₃O₄ -with metal oxides like CaO and Al₂O_3, which prevent reduction of the Fe with H₂) provides an absorptive surface to bring reagents together and facilitate bond breaking in H₂ and N₂.

 

• 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 nutrients for plants and perhaps the most important nutrients in regulating plant growth (primary productivity) and life diversity in the biosphere. All living organisms require feedstocks to produce energy and as substrates for biosynthetic reactions. Which ones are used depends on the organism. Plants are primary producers, so they use their synthesized carbohydrates for energy production and biosynthesis. For carnivores, proteins and their derived amino acids are the source of 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 are the source of energy. 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 starving conditions, the organisms' own proteins are broken down and used for oxidative energy production and for any remaining biosynthesis. In diseased states like diabetes, which can be likened to a starving state in the presence of abundant carbohydrates, both lipids and amino acids become sources of energy.

How are amino acids in animals oxidatively metabolized? Many pathways could be used to do so. 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. Ammonium is not oxidized to nitrite or nitrates in humans as it occurs in the soil by microorganisms. It can be recycled back into nucleotides or amino acids, eliminating excess amounts 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

This chapter broadens the scope of organic chemistry by emphasizing that life is not only based on carbon compounds but also relies heavily on nitrogen and oxygen. It presents a comprehensive overview of the biological nitrogen cycle, detailing both inorganic and organic transformations and highlighting the intricate interplay between microorganisms and the biosphere.

Key topics covered include:

• The Biological Nitrogen Cycle:
  The chapter outlines the major processes—nitrogen fixation, nitrification, denitrification, anammox, and ammonification—that convert atmospheric dinitrogen (N₂) into bioavailable forms such as ammonium (NH₄⁺), nitrite (NO₂⁻), and nitrate (NO₃⁻). These processes are carried out by specialized bacteria and archaea, both aerobically and anaerobically, ensuring that essential nitrogen is incorporated into biomolecules.

• Thermodynamics and Kinetics of Nitrogen Fixation:
  Although the conversion of N₂ to ammonia is thermodynamically favorable at room temperature (ΔG° < 0), the reaction is kinetically hindered due to the high stability of the N≡N triple bond. This necessitates the use of energy-intensive processes—such as the biological nitrogenase system in microbes or the industrial Haber-Bosch process—to overcome the activation energy barrier.

• Impact of Industrial Nitrogen Fixation:
  The chapter contrasts biological nitrogen fixation with the Haber-Bosch process, discussing how the latter has dramatically increased the amount of fixed nitrogen available globally, with significant ecological consequences such as nutrient runoff and eutrophication.

• Integration with Ecosystem Function:
  Nitrogen metabolites serve as crucial nutrients for plant growth, influencing primary productivity and ecosystem diversity. The chapter emphasizes that while plants synthesize carbohydrates for energy, animals rely on proteins and amino acids both as energy sources and for biosynthesis, making the efficient cycling of nitrogen vital for all life.

• Amino Acid Metabolism and Nitrogen Handling:
  In animals, the breakdown of proteins for energy requires the removal of the amino group to prevent toxic accumulation of ammonium. The resulting carbon skeletons are converted into ketoacids, which then enter central metabolic pathways like glycolysis and the citric acid cycle. The regulation of ammonium levels through recycling into biomolecules or elimination is crucial for maintaining metabolic balance.

Overall, the chapter illustrates how the study of the nitrogen cycle extends biochemistry beyond the confines of human metabolism to encompass broader ecological and environmental contexts. It sets the stage for further exploration of amino acid oxidation, emphasizing the necessity of understanding nitrogen metabolism to appreciate the full complexity of biological systems.