9.1 Nitrogen Fixation and Assimilation

Nitrogen fixation and assimilation are crucial processes in the nitrogen cycle. They convert atmospheric nitrogen into forms that living things can use, replenishing the pool of available nitrogen in ecosystems. Without these processes, life as we know it couldn't exist.

Microorganisms play a central role in nitrogen fixation, with some forming symbiotic relationships with plants. Plants and animals then assimilate this fixed nitrogen, using enzymes to incorporate it into biomolecules like proteins and DNA.

Nitrogen fixation: The process and its importance

Conversion of atmospheric nitrogen

Earth's atmosphere is about 78% nitrogen gas ($N_2$), but most organisms can't use it in that form. Nitrogen fixation converts $N_2$ into biologically available forms, primarily ammonia ($NH_3$) or ammonium ($NH_4^+$).

The reason this conversion is so energy-demanding is the triple bond in $N_2$. That bond is one of the strongest in nature, requiring specialized enzymes or extreme conditions to break.

• Fixation occurs through biological, industrial, or atmospheric processes
  • Biological fixation is the most significant in natural ecosystems
  • Industrial fixation uses the Haber-Bosch process (high temperature and pressure to synthesize $NH_3$ from $N_2$ and $H_2$)
  • Atmospheric fixation occurs via lightning, which provides enough energy to split $N_2$ and combine it with oxygen to form nitrogen oxides
• Once fixed, nitrogen can be used to synthesize amino acids, nucleic acids, and other nitrogen-containing biomolecules

Role in the nitrogen cycle

The nitrogen cycle describes the movement of nitrogen through the biosphere, atmosphere, and geosphere. Nitrogen fixation is the entry point for new biologically available nitrogen into this cycle.

• Without fixation, denitrification and other loss processes would gradually deplete the pool of available nitrogen
• That depletion would limit ecosystem productivity, since nitrogen is often the nutrient that constrains plant growth

Microorganisms: Key players in nitrogen conversion

Prokaryotic nitrogen fixers

Only certain prokaryotes (bacteria and archaea) possess the nitrogenase enzyme needed for biological nitrogen fixation. These organisms are collectively called diazotrophs, and they operate either as free-living organisms or in symbiotic partnerships with plants.

• Rhizobia form symbiotic relationships with leguminous plants (beans, peas, clover). They colonize specialized root nodules where conditions favor fixation. The plant supplies carbon to the bacteria; the bacteria supply fixed nitrogen to the plant.
• Frankia are actinobacteria that form similar symbiotic relationships with certain non-leguminous plants like alder trees. This is called actinorhizal symbiosis.
• Cyanobacteria fix nitrogen in aquatic ecosystems and some terrestrial environments. Some species use specialized cells called heterocysts to protect nitrogenase from oxygen.
• Free-living soil bacteria such as Azotobacter (aerobic) and Clostridium (anaerobic) also contribute to nitrogen fixation in soils, though typically at lower rates than symbiotic fixers.

Diverse nitrogen-fixing organisms

• Some archaea are capable of nitrogen fixation in extreme environments like hot springs, expanding the range of habitats where fixation occurs
• Symbiotic relationships enhance fixation efficiency because the plant host provides a protected, energy-rich environment for the microorganism

Nitrogen assimilation: From fixed to usable

Plant nitrogen assimilation

Plants take up nitrogen from the soil mainly as ammonium ($NH_4^+$) or nitrate ($NO_3^-$). What happens next depends on which form they absorb.

Ammonium assimilation is more direct. $NH_4^+$ can be incorporated into organic molecules without needing to be reduced first.

Nitrate assimilation requires a two-step reduction before the nitrogen can be used:

1. $NO_3^-$ is reduced to nitrite ($NO_2^-$) by the enzyme nitrate reductase
2. $NO_2^-$ is then reduced to $NH_4^+$ by nitrite reductase

Once in the $NH_4^+$ form, nitrogen enters the GS-GOGAT cycle, which is the primary pathway for incorporating nitrogen into amino acids:

1. Glutamine synthetase (GS) combines $NH_4^+$ with glutamate to form glutamine
2. Glutamate synthase (GOGAT) transfers the amide group from glutamine to $\alpha$-ketoglutarate, producing two molecules of glutamate

One of those glutamate molecules recycles back into step 1, while the other serves as a nitrogen donor. Transamination reactions, catalyzed by aminotransferases, then distribute nitrogen from glutamate to other amino acids throughout the plant.

Animal nitrogen assimilation and excretion

Animals can't fix or directly assimilate inorganic nitrogen. Instead, they obtain nitrogen by consuming plants or other animals and digesting the proteins and nucleic acids.

• Dietary proteins are broken down into amino acids, which are then reassembled into the animal's own proteins
• Excess nitrogen is excreted, typically as urea (mammals) or uric acid (birds, reptiles)
• Soil microorganisms further process these waste products, converting them back to $NH_4^+$ or $NO_3^-$, which reenters the nitrogen cycle

Enzymes: Catalysts for nitrogen fixation and assimilation

Nitrogen fixation enzymes

Nitrogenase is the enzyme complex responsible for biological nitrogen fixation. It has two main components:

• Dinitrogenase reductase (the iron protein) transfers electrons to the second component
• Dinitrogenase (the molybdenum-iron protein) uses those electrons to reduce $N_2$ to $NH_3$

The overall reaction requires a large energy input: at least 16 ATP per molecule of $N_2$ fixed.

A critical constraint is that nitrogenase is highly sensitive to oxygen, which irreversibly damages the enzyme. Organisms have evolved several strategies to deal with this:

• Leghemoglobin in legume root nodules binds free oxygen, keeping concentrations low enough to protect nitrogenase while still allowing aerobic respiration in the nodule cells
• Cyanobacteria use heterocysts, thick-walled cells that exclude oxygen
• Some free-living bacteria fix nitrogen only under anaerobic conditions

Nitrogen assimilation enzymes

• Nitrate reductase catalyzes $NO_3^- \rightarrow NO_2^-$ (the first step in nitrate assimilation)
• Nitrite reductase catalyzes $NO_2^- \rightarrow NH_4^+$ (completing the reduction)
• Glutamine synthetase (GS) incorporates $NH_4^+$ into glutamine, the key entry point for nitrogen into organic molecules
• Glutamate synthase (GOGAT) transfers the amide nitrogen from glutamine to $\alpha$-ketoglutarate, producing two glutamate molecules and completing the GS-GOGAT cycle

Together, these enzymes form a coordinated pathway that moves nitrogen from its inorganic, fixed form all the way into the amino acids that cells need to build proteins and nucleic acids.