16: Fixing Nitrogen Fixation

Nitrogen Fixation

We spent most of Chapter 22.1 discussing the biochemistry of nitrogenase, which fixes the stable molecule N₂ to form NH₃/NH₄⁺.  It's a highly complex reaction conducted by symbiotic microbes (prokaryotes) that fix N₂ for plants.  The world uses the Haber-Bosch process to produce over 100 million metric tons of nitrogen fertilizer that supports half of the world's population's food supply. Only about half of the ammonium added to soil is taken up by plants.  The rest is released into waterways or used by microbes, which can also produce it from the potent greenhouse gas nitrous oxide (N₂O).  It has a 300x greater effect than CO₂ based on weight. The oxidation number of N in NH₃  is -3, and in N₂O it is +1, showing that the NH₃ can be oxidatively metabolized 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 make nitrogenase a better and more efficient enzyme.  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 nitrogen fertilizer!

Genes of Nitrogen Fixation

The catalytic nitrogenase enzyme complex is encoded by three genes:

• 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).  With its partner nifD, it catalyzes the key enzymatic reactions as part of a heterodimer with nifD.  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 insufficient quantities of N₂ exist, the protein NtrC activates NifA expression, which activates the rest of the 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 in the folding of 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 2 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 are used to reduce each molecule of N₂.

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 the sensitivity of nitrogenase to O2.

Saccharomyces cerevisiae has been engineered using synthetic biology to express the NifDK nitrogenase tetrameric protein in their mitochondria (after post-translational import).  Yeast is a model organism, and tools have been developed for synthetic biology experiments using yeast. Much can be learned to be 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 the mitochondria, which again minimizes O₂ oxidative damage to the 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 was also able to reduce acetylene, HC=CH (an alternative substrate similar to N=N) after adding 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 mitochondria 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 in maximizing the expression of nitrogen in crop plants.

Synthetic Biology to Express Nitrogenase in Bacteria

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 the holobiont, which consists of the plant host and all species interacting 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 derivatives of γ-proteobacterium (KV137), found on corns roots and which can fix N₂.  They have been engineered to turn nitrogenase genes on when N₂ fixation is needed.  The engineered bacteria is added to liquid fertilizer, reducing the need for chemical fertilizer by 25 lb/acre) and at the same time, increasing yields.  This bacterial-based fertilizer does not wash into waterways with its negative environmental effects.  Likewise, no N₂O is produced on microbial metabolism of excess fertilizers, which decreases 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 sources of N₂. Rhizobia is one example that can fix N₂ in the nodules of legumes. The diazotroph isolated from corn roots and mentioned above, Kv137, was gene-edited to produce a modified strain (Kv137-1036) that fixes N₂ without inhibition by applied nitrogen fertilizers.

The Kv137 strain has nifA and nifL genes and proteins, which, as described above, regulate the expression of nitrogenase based on the need for nitrogen. These two genes are on one operon under the control of a single promoter.  nifL was replaced with another promoter, which removes the down-regulation of nifA since no nifL was present.  This allowed the expression and activity of nitrogen even in the presence of exogenous fertilizer. 

Figure \(\PageIndex{12}\) below shows that Kv137-1036 strain (red dots) does colonize 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)

Summary

This chapter provides an in‐depth exploration of biological nitrogen fixation—the enzymatic process by which certain prokaryotes convert atmospheric nitrogen (N₂) into ammonia (NH₃) or ammonium (NH₄⁺), making nitrogen accessible for plant growth. It contrasts the natural, enzyme‐catalyzed reaction with the industrial Haber–Bosch process, highlighting that while Haber–Bosch produces vast quantities of nitrogen fertilizer essential for global food production, only about half of this nitrogen is effectively used by plants. The surplus not only contributes to environmental issues such as waterway eutrophication but also to greenhouse gas emissions via microbial transformation into nitrous oxide (N₂O), a gas with a far greater warming potential than CO₂.

Central to biological nitrogen fixation is the nitrogenase enzyme complex, which comprises multiple protein subunits encoded by nif genes (nifH, nifD, and nifK). These subunits collaborate to facilitate the reduction of the inert N₂ molecule through a series of electron transfer reactions powered by ATP hydrolysis. The enzyme's activity relies on complex metalloclusters, including [4Fe–4S] clusters and the FeMo cofactor, which are essential for its catalytic function. In addition, several regulatory proteins (such as NifA, NifB, NifL, and NifM) orchestrate the expression, assembly, and activity of the nitrogenase complex in response to environmental cues like oxygen levels and available fixed nitrogen.

The chapter also discusses the potential of genetic engineering and synthetic biology to enhance biological nitrogen fixation. One promising approach involves the heterologous expression of nitrogenase components in non-leguminous crops (e.g., rice, corn, and wheat), potentially enabling these staple crops to self-supply nitrogen. Efforts in model organisms like Saccharomyces cerevisiae have demonstrated the feasibility of targeting nitrogenase expression to mitochondria, where lower oxygen levels and efficient iron-sulfur cluster assembly provide a more favorable environment for enzyme function.

Overall, the chapter emphasizes the critical role of biological nitrogen fixation in sustaining global primary production and the potential for innovative biotechnological interventions to reduce dependence on industrial nitrogen fertilizers, thereby mitigating environmental impacts and enhancing sustainable agriculture.