18.1: The Biochemistry of Nitrogen in the Biosphere

Search Fundamentals of Biochemistry

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. We'll concentrate on the metabolic fate of amino groups in amino acids and proteins in the next section. 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 both 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 ATPs. Ammonium once made can then be taken up by primary producers like plants and incorporated into biomolecules such as amino acids, which animals consume. For those who may still believe that people have marginal effects on our biosphere, consider this. We may soon fix more N₂ (to NH_3⁾ 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 both 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 genetically modify plants to make nitrogenase, eliminating the need for fertilizers but perhaps creating unforeseen problems of its own.

You might be surprised that at room temperature the equilibrium constant favors ammonia formation, 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 4 molecules to two molecules. From an enthalpy perspective, if you raise the temperature of an exothermic reaction, you drive it in a reverse direction. If you increase the pressure, you shift the equilibrium to the side with the fewest number of 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. Same with 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, but that would slow 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 that has fewer molecules of gas, 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 Al₂O₃ 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 the formation of nitrate (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 runoff water, polluting our rivers and lakes.

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

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

• Anammox reaction: This more 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 the regulation of plant growth (primary productivity) and in regulating 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 own synthesized carbohydrates for both 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 source of energy 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 their 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 in the unfed state, lipids take a predominant role. Under starving conditions, the organisms' own proteins are broken down and used for oxidative energy production and for any biosynthesis that remains. 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 the sources of energy.

How are amino acids in animals oxidatively metabolized? Many pathways could be used to do so but 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 occurs in the soil by microorganisms. It can be recycled back into nucleotides or amino acids, and excess amounts are eliminated from the organism. Both processes must be highly controlled. We will turn out attention to the oxidation of amino acids in the next section.