16: Fixing Nitrogen Fixation

Nitrogen Fixation

We spent most of Chapter 22.1 discussing the biochemistry of nitrogenase, which converts the stable molecule N₂ into NH₃/NH₄⁺.  It's a highly complex reaction conducted by symbiotic microbes (prokaryotes) that fix N₂ for plants.  Modern agriculture needs more than nitrogenase can provide.  The world uses the Haber-Bosch process to produce over 100 million metric tons of nitrogen fertilizer, which supports half of the world's food supply. Only about half of the ammonium added to the soil is taken up by plants.  The rest is released into waterways or used by microbes, which can convert it to the potent greenhouse gas nitrous oxide (N₂O).  It has a 300x greater effect per unit of climate-warming potential than CO₂. The oxidation number of N in NH₃  is -3, and in N₂O it is +1, indicating that NH₃ can be oxidized for energy production by the microbes. Also, excess NH₄⁺ goes into waterways and leads to eutrophication, the overproduction of algae and plankton, which depletes O₂ from the waters and kills other organisms.  

N₂O emissions have increased dramatically since 1850, as shown in the interactive graph from Our World in Data in Figure \(\PageIndex{9}\) below.

Figure \(\PageIndex{9}\):  Our World in Data. https://ourworldindata.org/grapher/n...ions?tab=chart

So, what can we do to "fix" nitrogen fixation to reduce reliance on the Haber process and its collateral climate effects?  Perhaps we could use mutagenesis to improve nitrogenase efficiency.  That would not be easy given the complexity of both the enzyme and the mechanism of N₂ conversion to NH₃, which requires many metal-ion cofactors.  A better alternative would be to express nitrogenase in plants so they could synthesize their own nitrogen fertilizer!  Also, maybe we can convert more food crops into species that can host nitrogen-fixing bacteria in their roots, forming a symbiotic relationship.  Then nitrogen can be fixed directly in the roots.

Before we discuss ways to "fix" nitrogen fixation, we will present a brief introduction to how all plants acquire fixed nitrogen.  Then we will explore ways to enhance that process to reduce the need for the Haber process and for fertilizers.

Recent Updates:  12/16/25

How do plants acquire fixed nitrogen?

All plants need fixed nitrogen, but none have nitrogenase. Seems like an evolutionary mismatch!  We presented nitrogenase in great detail in Chapter 22, but didn't mention that the enzyme can't function in the presence of much oxygen.  Aerobic plants don't have nitrogenase, so they must acquire fixed nitrogen elsewhere. How are these quandaries resolved?

All plants obtain fixed nitrogen from two sources: soil microbes that produce NH₄⁺, which can diffuse into plants, and leguminous plants that harbor symbiotic bacteria in root nodules, which produce NH₄⁺ within the plant.  Without microbes, no nitrogen would be fixed or made available to the plant. The bacteria in these nodules include the flagellated, Gram-negative, diazotrophic genus Rhizobia (rhiza means "root" in Greek, and bios means "life"). In this symbiotic relationship, rhizobia acquire carbohydrates and other nutrients from plants and, in turn, provide NH₄⁺ to them.  Figure \(\PageIndex{cc}\) below shows root nodules, which contain the nitrogen-fixing bacteria.

Figure \(\PageIndex{10}\): Root nodules containing rhizobia.  https://milnepublishing.geneseo.edu/...ter/rhizobium/.  

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The advantages to plants and the environment from using nitrogen-fixing bacteria in root nodules are many:

• They make their own NH₄⁺, so it doesn't have to diffuse through the soil from soil microbes
• The plant highly controls uptake
• Competition for NH₄⁺ by other species is eliminated
• Losses of NH₄⁺ are very low
• There are low levels of environmentally deleterious nitrogen products, including NO₃^- and N₂O.

Hence, producing fixed nitrogen in root nodules of legumes is highly efficient compared to non-leguminous plants.

Oxygen remains a potential problem because it can greatly interfere with nitrogenase in bacteria.  Nitrogenase is so sensitive to oxygen because it contains iron–sulfur clusters with relatively low reduction potentials. 

Let's review reduction potentials. Remember that O₂ has a standard reduction potential E^0' = 0.816 (see Table 12.4.2) and is a powerful oxidant, causing the loss of electrons in substrates that act as reducing agents for O₂ as it forms water.  Remember than ΔG^0' = -nFE⁰' for the reaction 1/2O₂ + 2H⁺ → H₂O.   

For the reaction of oxygen to form the reactive oxygen species, superoxide, instead of water (which doesn't require protons):

\begin{equation}
\mathrm{O}_2+\mathrm{e}^{-} \rightleftharpoons \mathrm{O}_2^{\bullet-}
\end{equation}

The standard reduction potential for superoxide formation (at pH 7) is about -0.33 V.  

When we consider enzymes with FeS centers, each has a different E^0' value, as the geometry and electrostatics at the FeS centers can vary widely.  We discussed this in Chapter 19.1: Electron-Transfer Reactions in Mitochondria.  

Instead of using E^0' values (analogous to pKa values for acid/base reaction), an alternative, E_m is often used for reactions involving complicated metal ion centers.  E_m is the midpoint reduction potential, measured not under standard conditions, but in a given set of experimental or physiological conditions.  It's analogous to pKa, which occurs at the midpoint of an acid/base titration curve. We know that pKas for proton-donating groups in proteins vary widely for different local environments. 

The approximate Em values for the FeS clusters of nitrogenase and the citric acid cycle enzyme aconitase (which also has an FeS cluster), compared to that of superoxide formation, are shown below:

• Nitrogenase FeS protein ~ −420 to −500 mV

• Aconitase ~ −200 to −350 mV

• O₂/O₂•⁻ ~ −330 mV

Comparing these negative values shows that the nitrogenase FeS centers are a stronger reductant than the FeS center in aconitase.  Let's do some quick calculations based on this equation from introductory chemistry.

\begin{equation}
\Delta G=-n F \Delta E
\end{equation}

Let's say the Em value of nitrogenase is around -0.5 V.  Using the equation:

\begin{equation}
\Delta E=E_{\text {acceptor }}-E_{\text {donor }}
\end{equation}

We can show that ΔE_nitrogenase ~ -0.33 V - (-0.5 V) = +0.17V so ΔG = -nFΔE = -16.4 kJ/mol or  -3.9 kcal/mol.  The same calculations for aconitase give a ΔG >0.  These quick calculations show that the FeS centers of nitrogenase readily react with O₂ to form O₂^- (superoxide), a reactive oxygen species.

In catalysis, these clusters spend significant time in reduced states (electron-loaded). O₂ can approach/bind weakly near exposed Fe sites or react to produce reactive oxygen species (see Chapter 12.3: The Chemistry and Biochemistry of Dioxygen).  These reactions are summarized below.

The bound O₂ can react with iron to form superoxide, as shown in the reaction below.

\begin{equation}
\mathrm{Fe}-\mathrm{S}(\text { reduced })+O_2 \rightarrow \mathrm{Fe}-\mathrm{S}(\text { oxidized })+O_2^{\bullet-}
\end{equation}

The superoxide can produce peroxides through a dismutation reaction without or with the enzyme superoxide dismutase to from peroxides as shown below:

\begin{equation}
2 \mathrm{O}_2^{\bullet-}+2 \mathrm{H}^{+} \rightarrow \mathrm{H}_2 \mathrm{O}_2+\mathrm{O}_2
\end{equation}

The peroxide can then react through the Fenton reaction to produce the hydroxy free radical as shown below:

\begin{equation}
\mathrm{Fe}^{2+}+\mathrm{H}_2 \mathrm{O}_2 \rightarrow \mathrm{Fe}^{3+}+\mathrm{OH}^{-}+\bullet \mathrm{OH}
\end{equation}

Locally produced ROS can oxidize sulfur and iron in nitrogenase, thereby inactivating it.  So O₂ doesn't just inhibit the enzyme, it destroys it, requiring new synthesis to replace it.  

Rhizobium is mainly aerobic in the soil outside of the root nodules, as it uses O₂ for respiration.  However, inside the nodules, it is functionally microaerobic since O₂ is so low.  Even then, O₂ is still a problem for N₂ fixation.  Rhizobia solve this oxygen problem through a protein called leghemoglobin (a better name for it would be legmyoglobin!).  This cytosolic protein acts as an O₂ buffer (much like myoglobin in skeletal and cardiac muscles) to keep free O₂ low and nitrogenase active. It also binds O₂ much more tightly than myoglobin. The P₅₀ of leghemoglobin is about 0.01-0.05 μM compared to 4-5 μM for myoglobin.  

Figure \(\PageIndex{2}\) shows an interactive iCn3D model showing the alignment of sperm whale myoglobin (1MBN) and soybean leghemoglobin (1BIN).  

Figure \(\PageIndex{2}\): Alignment of sperm whale myoglobin (1MBN, cyan) and soybean leghemoglobin (1BIN, magenta).  (Copyright; author via source). Click the image for a popup or use this external link: https://www.ncbi.nlm.nih.gov/Structu...6ec85ef13b1a77 

After viewing the initial alignment, toggle the "a" key to see each structure separately.  

Genes of Nitrogen Fixation - Can they be put into plants?

Inserting bacterial genes encoding nitrogenase into plants would allow them to produce their own NH₄⁺.  It's a complex endeavor, though.

Three genes encode the catalytic nitrogenase enzyme complex:

• nifH gene for the subunit Nitrogenase iron protein 1 (also called Nitrogenase component II, and Nitrogenase reductase).  It is a homodimer that binds one [4Fe-4S] cluster per dimer.
• nifD gene for the subunit Nitrogenase molybdenum-iron protein alpha chain (also called Dinitrogenase and Nitrogenase component I).  It catalyzes the key enzymatic reactions along with its partner nifK as part of a heterodimer. It binds one [8Fe-7S] cluster per heterodimer with nifK and 1 [7Fe-Mo-9S-C-homocitryl] cluster per subunit.
• nifK gene for the subunit Nitrogenase molybdenum-iron protein beta chain (also called Dinitrogenase and Nitrogenase component I).  In its heterodimeric partner, nifD, it catalyzes key enzymatic reactions.  It binds one [8Fe-7S] cluster per heterodimer with nifD and 1 [7Fe-Mo-9S-C-homocitryl] cluster per subunit.

The nitrogenase enzyme complex has regulatory proteins as well:

• nifA - Nif-specific regulatory protein required for activation of most nif operons.  It senses N₂.  If N₂ is insufficient, the protein NtrC activates NifA expression, which in turn activates the remaining genes.
• nifB - FeMo cofactor biosynthesis protein NifB (also called FeMo-cofactor maturase NifB, Nitrogenase cofactor maturase NifB, and radical SAM assemblase NifB).  
• nifL - Nitrogen fixation regulatory protein required for inhibiting NifA activity (i.e., nitrogenase formation) in response to oxygen and low levels of fixed nitrogen.
• nifE - Periplasmic [NiFe] hydrogenase small subunit.
• NifM - a possible peptidyl-prolyl cis‐trans isomerase (i.e., a protein chaperone) that helps fold NifH.

Figure \(\PageIndex{10}\) below summarizes these gene products.

Figure \(\PageIndex{10}\): Minimum set of nif genes essential for nitrogen fixation with molybdenum-iron nitrogenase.  EMILY M. BENNETT et al., BIODESIGN RESEARCH.  10 Jan 2023, Vol 5, https://spj.science.org/doi/10.34133/bdr.0005 .  Creative Commons Attribution License 4.0 (CC BY 4.0).https://doi.org/10.34133/bdr.0005

The stoichiometry depicted has not been adjusted. NifB contains one catalytic cluster (shown in white) and two substrate [4Fe-4S] clusters that react to produce the NifB cofactor. NifEN matures the NifB cofactor, producing the FeMo cofactor. The molybdenum-iron (MoFe) nitrogenase (NifHDK) contains the FeMo cofactor at its active site. Electron donors transfer single electrons to the [4Fe-4S] cluster at the interface of the NifH homodimer. Electrons are moved from the [4Fe-4S] cluster into the active site of nitrogenase using energy produced by ATP hydrolysis by NifH. A minimum of 8 electrons is required to reduce each N₂ molecule.

It would be especially important to express nitrogenase in the main cereal food crops (rice, corn, and wheat), which get their nitrogen from soil microbes (in contrast to legumes, which contain nitrogen-fixing bacteria in nodules in their roots). It's a daunting task given the complexity of the protein complex, its metal cofactors, their inhibition by O₂, and the multiple genes required for nitrogenase regulation. Ideally, the relevant gene clusters could be moved into a chloroplast, which is evolutionarily derived from bacteria, so the gene regulation system might be more suitable. It also has low O₂ levels at night.  However, O₂ is produced in chloroplasts, which is problematic given nitrogenase's sensitivity to O₂.

Saccharomyces cerevisiae has been engineered using synthetic biology to express the NifDK nitrogenase tetramer in its mitochondria (via post-translational import).  Yeast is a model organism, and tools have been developed for synthetic biology experiments with it. Much can be learned and applied to other eukaryotic organisms, such as plants.  Mitochondria have high O₂ consumption (as opposed to production as in the chloroplast) and the ability to synthesize bacterial-type iron-sulfur clusters.  The Nif gene clusters were engineered into the XV chromosome as shown below in Figure \(\PageIndex{11}\).  

Figure \(\PageIndex{11}\): nif gene assembly in yeast. Buren et al., ACS Synth. Biol. 2017, 6, 6, 1043–1055. https://doi.org/10.1021/acssynbio.6b00371.  CC-BY license

Panel (a) shows the assembly strategy for transcription units, subclusters, and full clusters inserted by homologous recombination in the genome of S. cerevisiae. Panel (d) shows a diagram of nif gene organization in DSN14, a strain of S. cerevisiae.

Nitrogenase activity has also been functionally expressed in transgenic rice containing the NifH with a [4Fe-4S] cluster from Hydrogenobacter thermophilus and NifM (a peptidyl-prolyl cis‐trans isomerase from Azotobacter vinelandii), which helps NifH fold.  They were correctly targeted to mitochondria, thereby minimizing O₂-induced oxidative damage to metal-ion cofactors. The purified protein could transfer electrons to the MoFe protein (NfiDK dimer), but did so poorly.  It also assisted in the assembly of the FeMo cofactor.  However, the [4Fe-4S] cluster occupancy in the protein was poor. However, the purified protein could also reduce acetylene, HC=CH (an alternative substrate similar to N=N), upon addition of purified NifDK. 

Many steps must be optimized to create a functional nitrogenase in rice, corn, and wheat plants.  For example, mitochondrial-expressed NifD is readily cleaved by a mitochondrial endoprotease. Some NifD subunits are more resistant to proteolysis, and a single amino acid change (Y100Q) leads to enhanced protein stability.  AI will likely be extremely useful for maximizing nitrogen use efficiency in crop plants.

Interaction of nitrogen-fixing bacteria with nonleguminous plants

Bacteria can express nitrogenase that can fix atmospheric N₂. Still, they won't work with the critical cereal crops unless the bacteria can interact with roots in the "rhizosphere", the layers of dirt intimately in contact with roots. This life in this area is often called a holobiont, which consists of the plant host and all species that interact with it in a symbiotic relationship.  The metabolism in the holobiont is complex.  For example, plant carbohydrates are used by other organisms in the holobiont. It's similar to the gut biome, which consists of an ecosystem of human and microbial cells.

Bacteria have now been engineered to express nitrogenase AND interact with corn roots to fix N₂.  The cells are derived from a γ-proteobacterium (KV137) found on corn roots that fixes N₂.  They have been engineered to turn nitrogenase genes on when N₂ fixation is needed.  The engineered bacteria are added to liquid fertilizer, reducing the need for chemical fertilizer by 25 lb/acre while increasing yields.  This bacterial-based fertilizer does not wash into waterways with its negative environmental effects.  Likewise, no N₂O is produced during microbial metabolism of excess fertilizers, thereby decreasing the release of this potent greenhouse gas. In 2021, it was used on 3 million acres of corn.

One problem with using biological N₂ microbes in agricultural settings is that high levels of chemical fertilizers can effectively inhibit microbial N₂ fixation, which is regulated (as mentioned above) to shut down if nitrogen is bioavailable.  Still, bacteria that fix N₂ can produce up to 10% of the nitrogen requirement.  Genetic engineering is needed to overcome this inhibition.  Such bacteria are diazotrophs, as they can fix N2 and grow without exogenous N₂ sources. Rhizobia is one example of a bacterium that can fix N₂ in legume nodules. The diazotroph isolated from corn roots and described above, Kv137, was gene-edited to produce a modified strain (Kv137-1036) that fixes N2 without being inhibited by applied nitrogen fertilizers.

The Kv137 strain has the nifA and nifL genes and proteins, which, as described above, regulate nitrogenase expression in response to nitrogen availability. These two genes are on one operon under the control of a single promoter.  nifL was replaced with another promoter, thereby removing the nifA downregulation, since nifL was absent.  This allowed nitrogenase to be expressed and active even in the presence of exogenous fertilizer. 

Figure \(\PageIndex{12}\) below shows that the Kv137-1036 strain (red dots) colonizes corn roots.

Figure \(\PageIndex{12}\): Commercial efficacy of strain Kv137-1036 - Colonization of corn roots by microbes (red) after germination.  Wen et al., Enabling Biological Nitrogen Fixation for Cereal Crops in Fertilized Fields.  ACS Synth. Biol. 2021, 10, 12, 3264–3277. December 2021.  https://doi.org/10.1021/acssynbio.1c00049.  Attribution 4.0 International (CC BY 4.0)

Recent Updates:  1/6/26

Controlling Plant Immune Response to Allow Symbiosis and Nitrogen Fixation

Another approach to enable plants to synthesize fixed nitrogen is to allow them to harbor nitrogen-fixing bacteria in their root hairs (as in legumes).  This would require engineering the plant to take up soil bacteria to enable a symbiotic relationship, while also disabling any plant immune response to the symbiotic pathogen.  A detailed knowledge of the signaling pathways for bacterial uptake and the plant's immune response is required.

Signaling molecules are required for both symbiosis and an immune response.  Lipo-chitooligosaccharides (LCOs), most commonly known in agriculture and biology as Nod factors (Nodulation factors), are complex signaling molecules used by soil bacteria and fungi to communicate with plant roots.   An example of an LCO is shown in Figure \(\PageIndex{xx}\) below.

Figure \(\PageIndex{xx}\):  An example of an Lipo-chitooligosaccharides (LCOs) 

It is a β 1,4-linked polymer of N-acetylglucosamine (GlcNAc, blue ring).  An elongated polymer of β-1,4-linked N-acetylglucosamine, known as chitin (which we explored in Chapter 7.2: Polysaccharides), is found in insect shells and fungal cell walls.  In this particular LCO, an 18:1^Δ11 fatty acid in amide replaces the usual acetyl group.

LCOs are signaling molecules produced primarily by two types of soil microorganisms:

• Rhizobia Bacteria: These nitrogen-fixing bacteria release LCOs called Nod factors that are recognized by legumes and lead to root nodule formation; 
• Mycorrhizal Fungi: These fungi produce LCO-like Myc factors that allow plants to absorb phosphorus through a symbiotic relationship with the plant.  

Specificity in plant root recognition and the ultimate uptake of specific bacteria into plant roots is based on the length of their chitin and lipid chains and on signaling processes that follow. 

A key protein in the immune response towards LCOs and other chitin derivatives is the Lysin motif (LysM) receptor kinase.  It is a monotopic (single membrane pass) protein with an intracellular Ser/Thr protein kinase domain and an extracellular mltD domain which binds chitin derivative.  It acts as a pathogen-associated molecular pattern (PAMP) receptor.  (We discussed PAMPs in Chapter 5.04: B. The Innate Immune System, PAMPs and DAMPs, and Inflammation). The PAMP in this case is the LCO.  The chitin PAMP is recognized by lysine motif (LysM) domains in the extracellular mltD domain of the receptor kinase. 

Figure \(\PageIndex{xx}\) shows an interactive iCn3D model of the ectodomain (extracellular) of the cell surface receptor chitin elicitor receptor kinase 1 (CERK1) of the plant Arabidopsis bound to (NAG)₄, a short chitin fragment (4EBZ).  

Figure \(\PageIndex{xx}\): Ectodomain of the cell surface receptor chitin elicitor receptor kinase 1 of Arabidopsis bound to (NAG)₄ (4EBZ).  (Copyright; author via source). Click the image for a popup or use this external link: https://www.ncbi.nlm.nih.gov/Structu...47cfed55bec523

The ecto (extracellular) domain has three lysine motifs (LysM1-LysM3) domains (cyan, magenta, and gold), that can bind chitin.  In the above model, only the magenta LysM2 interacts with a short chitin chain, (NAG)₄.  Side chains in LysM2 that are involved in binding are shown as sticks and labeled.

Figure \(\PageIndex{xx}\) shows an interactive iCn3D model of the AlphaFold predicted structure of the Arabidopsis chitin elicitor receptor kinase 1(CERK1) involved in the immune response (CERK1, A8R7E6 · CERK1_ARATH)to pathogens (fungi, bacteria)  

Figure \(\PageIndex{xx}\): AlphaFold model of the Arabidopsis chitin elicitor receptor kinase 1 (CERK1, A8R7E6 · CERK1_ARATH).  (Copyright; author via source). Click the image for a popup or use this external link: https://www.ncbi.nlm.nih.gov/Structu...db9d41e1f20f2d

The disconnect between the intracellular part of the transmembrane helix and the kinase domain results from a poor AlphaFold-predicted structure for amino acids Ser 262 to Lys 306.  If larger chitin fragments are used (e.g., an octamer), the protein dimerizes. 

This PAMP receptor, when bound to a chitin derivative, induces innate immune signaling and resistance to pathogenic fungi. However, the same receptor also facilitates symbiosis with arbuscular mycorrhizal fungi.  This soil fungus lives in symbiosis with most land plants.  They enter roots and create arbuscules (tree-like structures) instead of root nodules like those found in legumes.  Images of a arbuscular mycorrhizal fungus in colonizing roots are shown in Figure \(\PageIndex{xx}\) below.

Figure \(\PageIndex{xx}\):  Arbuscular mycorrhizal fungus colonizing roots.  Left:   Arbuscule of Rhizophagus irregularis colonizing a maize root observed under confocal microscope.  Hector Montero.  WikiCommons.  https://commons.wikimedia.org/wiki/F...AArbuscule.png.  Middle:   Root tuber colonized by an arbuscular mycorrhizal fungus.  WikiCommons.  https://commons.wikimedia.org/wiki/F...ce=chatgpt.com.  Right:  Microscopic image of arbuscular mycorrhiza. MS Turmel, University of Manitoba, Plant Science Department, Public Domain, https://commons.wikimedia.org/w/inde...?curid=7553044. 

The root hairs on a root are not the fungi, as the fungal hyphae are too fine to see without a microscope unless they are clumped together.  The fungi are mostly inside the root and appear like a little tree (left and right image above).  They are not visible as hairs, but the hyphae do extend into the soil (middle image).  Arbuscules facilitate nutrient exchange and increase the transfer of water, phosphorus, and nitrogen into plants, while plants send fixed carbon to the fungus.

Resistance to such fungi in legume root cells must be overcome for symbiosis to occur via immune tolerance.  The symbiotic pathway in legumes is activated by Nod receptors. The CERK6 receptor, involved in an immune response, and the Nod Factor Receptor 1 (NFR1), involved in symbiosis, are highly similar in structure across their extracellular and intracellular domains, as well as in their interactions with chitin ligands.  

The NFR1 receptor (for symbiosis) from Lotus japonicus has a LysM receptor-like kinase that binds Nod factors from rhizobial bacteria. The intracellular part of the Nod factor receptor one (NFR1) can be mutated at two residues (T304M and D306A) to shift signaling from an immune response to a symbiotic response.  These mutations occur in the Symbiosis Determinant 1, located in the juxtamembrane section, a flexible region of 40 or more amino acids between the C-terminal part of the transmembrane helix and the kinase domain. 

Comparing the structures of CERK6 and an ortholog of NFR1, LYK3, shows that symbiosis determinant 1 is exposed internally in the cell and can mediate protein:protein interactions and hence signaling (i.e., not binding to the chitin ligand).  If the symbiosis determinant 1 sequence of NFR1 is swapped into the immune receptor CERK6, it is converted into one that allows bacterial infection and the formation of root nodules, even in non-legume plant receptors (such as barley)! 

These experiments suggest a way in which nitrogen-fixing symbiosis evolved from earlier immune receptors.

The LysM receptor kinase from Medicago is an ortholog of NFR1 from Lotus japonicus.  It is a chitin-ligand-symbiosis receptor and contains Met and Ala at positions 303 and 305, respectively.   Figure \(\PageIndex{xx}\) shows an interactive iCn3D model of the Medicago Truncatula LYK3 kinase domain with M304 and A306  (9GFZ).

Figure \(\PageIndex{xx}\): Medicago Truncatula LYK3 kinase domain with M304 and A306  (9GFZ).  (Copyright; author via source). Click the image for a popup or use this external link: https://www.ncbi.nlm.nih.gov/Structu...83af4acd1333b1

Summary

(Summary written by Claude, Anthropic)

The Haber-Bosch process produces over 100 million metric tons of nitrogen fertilizer annually — supporting roughly half of the world's food supply — but only about half of this applied nitrogen reaches plants. The rest enters waterways (causing eutrophication and O₂ depletion) or is converted by soil microbes to nitrous oxide (N₂O), a greenhouse gas with 300 times the warming potential of CO₂ per unit mass. N₂O emissions have risen dramatically since 1850. This chapter explores three strategies to reduce dependence on the Haber process by improving or extending biological nitrogen fixation.

Why nitrogenase is O₂-sensitive. Nitrogenase contains iron-sulfur clusters with very negative midpoint reduction potentials (Em ≈ −420 to −500 mV), making them thermodynamically spontaneous reductants of O₂ (Em for O₂/O₂•⁻ ≈ −330 mV; ΔG ≈ −16.4 kJ/mol). The resulting superoxide dismutates to H₂O₂, which generates the hydroxyl radical via the Fenton reaction, oxidizing and destroying the enzyme's FeS centers. Unlike reversible inhibition, O₂ destroys nitrogenase outright, requiring new enzyme synthesis.

The root-nodule solution. Legumes solve the O₂ problem by housing rhizobial bacteria in root nodules, where the protein leghemoglobin buffers free O₂ at extraordinarily low levels. Its P50 (≈0.01–0.05 μM) is 100–500 times tighter than myoglobin (≈4–5 μM), keeping the nodule interior near-anoxic while still supplying O₂ for rhizobial respiration. This symbiosis delivers NH₄⁺ directly within the plant with minimal N₂O production, no soil runoff, and tight control of nitrogen uptake — advantages that make it far more efficient than relying on soil microbes.

Engineering nitrogenase into crop plants. Transferring nitrogen fixation into cereal crops requires at minimum three structural genes (nifH, nifD, nifK) encoding subunits with elaborate FeS and FeMo cofactors, plus regulatory and maturation genes (nifA, nifB, nifL, nifM). Mitochondria are preferred over chloroplasts as a target organelle because they consume O₂ rather than producing it, and they can synthesize bacterial-type FeS clusters. The full nif gene cluster has been expressed in S. cerevisiae mitochondria (assembled by homologous recombination on chromosome XV). In transgenic rice, NifH targeted to mitochondria can transfer electrons to purified NifDK and reduce acetylene (an N₂ analog). Key obstacles remain: FeS cluster occupancy is poor, mitochondrial proteases cleave NifD (a Y100Q mutation improves stability), and full N₂ reduction in planta has not yet been achieved.

Engineering root-colonizing bacteria. A complementary approach uses naturally occurring diazotrophic bacteria. The corn-root bacterium Kv137 was engineered into strain Kv137-1036 by replacing the nifL gene (which normally shuts down nitrogenase when fixed nitrogen is available) with an independent promoter, thereby uncoupling nitrogenase expression from nitrogen availability. The strain colonizes corn roots, reduces chemical fertilizer requirements by ~25 lb/acre, increases yield, produces no N₂O, and causes no waterway runoff. By 2021 it was deployed on 3 million acres of corn — a real-world demonstration of synthetic biology improving agricultural sustainability.

Controlling plant immune responses to enable symbiosis. A third strategy — engineering non-legume plants to accept nitrogen-fixing bacteria directly into their root hairs — requires understanding how plants distinguish symbiotic microbes from pathogens. Both relationships are initiated by lipo-chitooligosaccharides (LCOs): Nod factors from rhizobia and Myc factors from mycorrhizal fungi, recognized by LysM receptor kinases on root cells. The immune receptor CERK1 and the symbiosis receptor NFR1 are structurally similar and bind the same chitin-based ligands; the difference between immune and symbiotic outcomes is determined by a short juxtamembrane region called Symbiosis Determinant 1. Two point mutations in this region of NFR1 (T304M, D306A) shift signaling from immune defense to symbiotic acceptance. Swapping the Symbiosis Determinant 1 sequence from NFR1 into CERK6 converts it into a symbiosis receptor that enables root nodule formation even in non-legumes such as barley — suggesting that nitrogen-fixing symbiosis evolved from ancestral immune receptors through modification of intracellular signaling, and pointing toward a tractable path for engineering this capability into cereal crops.