# Biochemistry of Biological Nitrogen Fixation

Ajeet Kumar

► To cite this version:

Ajeet Kumar. Biochemistry of Biological Nitrogen Fixation. Master. Biopesticide and Biofertilizer (Elective course), Dr. Rajendra Prasad Central Agricultural University, Samastipur, India. 2025, pp.1-21. hal-05267052

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<figure><img src="image_1.png" /></figure>

Distributed under a Creative Commons Attribution 4.0 International License
Dr. Rajendra Prasad Central Agricultural University

Pusa (Samastipur)-Bihar- 848125

A study Material

On

Lecture title: Biochemistry of Biological
Nitrogen Fixation

<figure><img src="image_2.png" /></figure>

COURSE TITLE: Bio-pesticides and Bio-fertilizers

*(Elective course)

COURSE NO.: ASS-307

CREDIT HOUR: 3 (2+1)

YEAR/SEMESTER: 3rd YEAR / 6th SEMESTER

Dr. AJEET KUMAR

Assistant Professor-cum-Scientist

Department of Microbiology

Collage of Basic Sciences and Humanities (CBS&H), Dr. RPCAU, Pusa

2025
# Biochemistry of Biological Nitrogen Fixation

Dr. AJEET KUMAR<fnref n="1" />

Nitrogen is an essential element for plant growth and development and a key issue of agriculture. Nitrogen fertilizers contribute to resolving the challenge the world is facing, feeding the human population. The Green Revolution was accompanied by an enormous increase in the application of nitrogen fertilizer. There is however a high heterogeneity of its distribution throughout the world. Biological Nitrogen Fixation (BNF) is known to be a key to sustain agriculture and to reduce soil fertility decline.

## Nodulation: From the Infection Process to the Functioning of the Nitrogenase

The production of nitrogen fertilizer by industrial fixation generates large quantities of carbon dioxide, contributing to earth warming. The natural process of BNF offers an economic means of reducing environmental problems and improving the internal resources. It is a process that allows microorganisms to convert atmospheric nitrogen ($N_2$) to ammonia ($NH_3$) assimilable by associated plants.

$$6 \text{ N} + 6\text{H}^+ + \text{ATP} \xrightarrow{6e^-} 2 \text{ NH}_3 + \text{ADP} + \text{Pi}$$

Different types of associations are listed as bellows:

<table><thead><tr><th>Types of association</th><th>Microorganisms</th><th>Host plants</th></tr></thead><tbody><tr><td rowspan="3">Symbiotic</td><td>Bacteria(ex. Rhizobium)<sup>a</sup></td><td>Legumes</td></tr><tr><td>Actinomycetes (ex. Frankia)</td><td>Actinorhiza</td></tr><tr><td>Cyanobacteria (ex. Anabaena azollaea)</td><td>Fern</td></tr><tr><td>Non symbiotic</td><td>Bacteria (ex. Azotobacter, Azospirillum)</td><td>Cereals</td></tr><tr><td>Free living systems</td><td>Bacteria (ex. Thiobacillus, Clostridium)</td><td></td></tr></tbody></table>

1. Allorhizobium (fast growing)

2. Azorhizobium (fast growing)

3. Bradyrhizobium (slow growing)

4. Mesorhizobium (intermediate growing)

5. Rhizobium (fast growing)

6. Sinorhizobium (fast growing)

<footnote>a: Rhizobium-leguminous plants is the most studied symbiotic association. There are six main genus of rhizobia:</footnote>

<footnote n="1">¹ Assistant Professor-cum-Scientist; Department of Microbiology, Collage of Basic Sciences and Humanities (CBS&H), RPCAU, Pusa (Samastipur)-848125, Bihar
E-mail: ajeetrau@gmail.com; ajeetrpcau@gmail.com</footnote>
## Nodule formation:

Leguminous plants and rhizobia communicate through the gene expression by reciprocally transmitting signals for the activation of the symbiotic genes in two partners. A type of phenolic called flavonoids, are released by host roots plants into the rhizosphere. Flavonoids act as a chemo-attractant for the bacteria to the plant roots, and eventually colonies of rhizobia attach to the root hairs. Flavonoid signal activates expression of nodulation (nod) genes.

In the rhizobial strains there are numerous nodulation genes including the nod genes nodABC and nodD (Figure 1). On the surface of the rhizobial bacteria the flavonoids are recognized by a expressed nodD protein. Then nodD binds to a promoter DNA sequence, and thereby activates transcription of nod genes of the operons. A group of nod genes encode enzymes synthesise the rhizobial nodulation signal, Nod factor (Figure 2), which triggers development of the root nodules by the plant. The plant roots recognise Nod factor, through binding to a surface protein receptor at the sub apical root tip. Perception of Nod factor induces a development inside the root, producing pronounced curling of the root hairs entrapping the rhizobia which establish infection of root. Bacteria gain access to plants cell membrane. The plasma membrane invaginates to form novel infection structure known as the infection thread, a tubular structure that extends from the root hair tip to the lower cells of the root cortex. Rhizobia enter the infection threads in which they actively multiply. At the same time the underlying root cortex cells are quickly proliferated to constitute the nodule primordia. The infection threads branch out into cells of the nodule primordia. The rhizobia are finally released into the nodule cell and enveloped in a membrane derived from the host cell plasma membrane. At this stage, rhizobial bacteria become bacteroids able to fix nitrogen.

## Importance of nitrogen fixation

The process of nitrogen fixation is carried out in microbes.

## Key Points

* Nitrogen fixation takes elemental nitrogen ($N_2$) and converts it into a ammonia, a format usable by biological organism.

* The fixed form of nitrogen ($NH_3$) is needed as an essential component of DNA and proteins. Therefore, it is needed for all life on earth.

* Nitrogen fixation is carried out by the enzyme nitrogenase, which are found in microbes.
## Key Terms

* **Fixation**: The act of uniting chemically with a solid substance or in a solid form; reduction to a non-volatile condition; — said of gaseous elements.

* **cofactor**: A substance, especially a coenzyme or a metal, that must be present for an enzyme to function.

* **Heterometal**: Describing a complex containing two (or more) different metals

* **symbiosis**: A close, prolonged association between two or more organisms of different species, regardless of benefit to the members

* **sulfide**: Any compound of sulfur and a metal or other electropositive element or group.

* **reduction**: A reaction in which electrons are gained and valence is reduced; often by the removal of oxygen or the addition of hydrogen.

* **enthalpy**: In thermodynamics, a **measure of the heat content of a chemical** or physical system.

* **oxidation**: A reaction in which the atoms of an element lose electrons and the valence of the element increases.

* **reduction**: A reaction in which electrons are gained and valence is reduced; often by the removal of oxygen or the addition of hydrogen.

* **proteoglycan**: Any of many glycoproteins that have heteropolysaccharide side chains.

## Nitrogen fixation:

The conversion of atmospheric nitrogen into ammonia and organic derivatives, by natural means, especially such conversion, by microorganisms in the soil, into a form that can be assimilated by plants.

Nitrogen fixation also refers to other biological conversions of nitrogen, such as its conversion to nitrogen dioxide. Nitrogen fixation is a process by which nitrogen ($N_2$) in the atmosphere is converted into ammonia ($NH_3$). Atmospheric nitrogen or elemental nitrogen ($N_2$) is relatively inert: it does not easily react with other chemicals to form new compounds. Dinitrogen is quite inert because of the strength of its $N \equiv N$ triple bond. To break one nitrogen atom away from another requires breaking all three of these chemical bonds. Fixation processes free up the nitrogen atoms from their diatomic form ($N_2$) to be used in other ways. Nitrogen fixation, natural and synthetic, is essential for all forms of life because nitrogen is required to biosynthesize basic building blocks of plants, animals, and other life forms, e.g., nucleotides for DNA and RNA and amino acids for proteins. Therefore, nitrogen fixation is
essential for agriculture and the manufacture of fertilizer. Microorganisms that fix nitrogen are bacteria called diazotrophs.

## Nitrogenase and Nitrogen Fixation

Some higher plants, and some animals (termites), have formed associations (symbioses) with diazotrophs. Diazotrophs are microbes. Biological nitrogen fixation was discovered by the German agronomist *Hermann Hellriegel* and Dutch microbiologist Martinus Beijerinck.

Biological nitrogen fixation (BNF) occurs when atmospheric nitrogen is converted to ammonia by an enzyme called nitrogenase. Nitrogenases are enzymes used by organisms to fix atmospheric nitrogen gas ($N_2$). There is only one known family of enzymes that accomplishes this process. All nitrogenases have an iron – and sulfur-containing cofactor that includes a heterometal complex in the active site (e.g., FeMoCo). In most species, this heterometal complex has a central molybdenum atom. However, in some species it is replaced by a vanadium or iron atom. Enzymes responsible for nitrogenase action are very susceptible to destruction by oxygen. Many bacteria cease production of the enzyme in the presence of oxygen. Many nitrogen-fixing organisms exist only in anaerobic conditions, respiring to draw down oxygen levels, or binding the oxygen with proteins.

## History of Nitrogen Fixation

For thousands of years farmers were aware that plants belonging to the legume family, promoted crop growth when planted with other non-legumes. Growing a legume crop in a field could also result in the next year's crop of non-legume plants giving a far greater yield. This led to the practice of crop rotation, a practice which can be traced back to techniques recorded in Roman literature.

<figure><img src="image_3.png" /></figure>

**Martinus Beijerinck:** Work done by Martinus Beijerinck was key to the discovery of rhizobia, symbiotic bacteria found on the roots of legumes, responsible for nitrogen fixation.

While the ancient Romans were aware of the improved results gained through crop rotation, they did not know that these benefits were brought about through the replenishment of nitrogen in the soil. Later people knew legumes did replenish nitrogen in the soil, but did not know how atmospheric ($N_2$) was converted into ammonium ($NH_3$) by legumes until research done in the 19th century.

*Hermann Hellriegel (1831-1895)*, a noted German agricultural chemist, discovered that leguminous plants took atmospheric nitrogen and replenished the ammonium in the soil
through the process now known as nitrogen fixation. He found that the nodules on the roots of legumes are the location where nitrogen fixation takes place.

## HELLRIEGEL'S AND BEIJERINCK'S DISCOVERIES

Hellriegel did not determine what factors in the root nodules carried out nitrogen fixation. Martinus Willem Beijerinck (March 16, 1851 – January 1, 1931), a Dutch microbiologist and botanist, explored the mechanism responsible, discovering that the root nodules contained microbes. He further demonstrated that these microbes were bacteria, which he named rhizobia. These rhizobia perform the chemical processes of nitrogen fixation. In addition to having discovered this biochemical reaction vital to soil fertility and agriculture, Beijerinck is responsible for the discovery of this classic example of symbiosis between plants and bacteria. The bacteria in the root nodules are needed to provide nitrogen for legume growth, while the rhizobia are dependent on the root nodules as an environment to grow.

## Nitrogen Fixation Mechanism

The conversion of N₂ to NH₃ depends on a complex reaction, essential to which are enzymes known as nitrogenases.

### Key Points

* ✓ Nitrogen fixation does result in the release of energy, but the activation of this reaction takes energy in the form of ATP hydrolysis.

* ✓ Nitrogenases are metalloenzymes, which are proteins that have metallic molecules as subunits.

* ✓ While a great deal is known about how nitrogenases reduce nitrogen, some steps are unknown.
## Biological nitrogen fixation (BNF)

It occurs when atmospheric nitrogen is converted to ammonia by an enzyme called nitrogenase. The reaction for BNF is:

$$N_2 + 8 H^+ + 8 e^- \rightarrow 2 NH_3 + H_2$$

This type of reaction results in N₂ gaining electrons and is thus termed a reduction reaction. While the equilibrium formation of ammonia from molecular hydrogen and nitrogen has an overall negative enthalpy of reaction (i.e. it gives off energy), the energy barrier to activation is very high without the assistance of catalysis, which is done by nitrogenases. The enzymatic reduction of N₂ to ammonia therefore requires an input of chemical energy, released from ATP hydrolysis, to overcome the activation energy barrier.

### A General Catalytic Mechanism Scheme for Nitrogenase:

a) Components I and II are dissociated; II is ready for reduction.

b) ATP binds to component II, which receives electrons from an electron donor (ferredoxin or flavodoxin); binding of ATP induces an allosteric conformational change which allows association of the two proteins. Electrons flow from the [4Fe-4S] cluster on II to the P cluster on I.

c) Electrons are further shuttled to the iron-molybdenum cofactor (FeMoco), and ATP is hydrolised to ADP. This step is repeated several times before a molecule of N₂ can bind to FeMoco.

d) The protein complex dissociates, and nitrogenase reduces dinitrogen to ammonia and dihydrogen.

(Legend: I: component I (dinitrogenase; MoFe protein); II: component II (dinitrogen reductase; Fe protein); ATP: adenosine triphosphate; ADP: adenosine diphosphate; Fdx: ferredoxin; Fld: flavodoxin).

Nitrogenase is made up of two soluble proteins: component I and II. Component I known as MoFe protein or nitrogenase contains 2 Mo atoms, 28 to 34 Fe atoms, and 26 to 28 acid-labile sulfides, also known as a iron-molybdenum cofactor (FeMoco). Component I is composed of two copies each of two subunits (α and β); each subunit's stability depends on the other in vivo.

Component II known as Fe protein or nitrogenase reductase is composed of two copies of a single subunit. This protein has four non-heme Fe atoms and four acid-labile sulfides (4Fe-4S). Substrate binding and reduction takes place on component I, which binds to ATP and ferredoxin or flavodoxin proteins (Fdx or Fld) (see step B).

The hydrolysis of ATP supplies the energy for the reaction while the Fdx/Fld proteins supply the electrons. Note this is a reduction reaction which means that electrons must be Page 7 of 21
added to the N₂ to reduce it to NH₄. Thus, the role of component II is to supply electrons, one at a time to component I. ATP is not hydrolyzed to ADP until component II transfers an electron to component I. The association of nitrogenase component I and II and later dissociation occurs several times to allow the fixation of one N₂ molecule.

Nitrogenase ultimately bonds each atom of nitrogen to three hydrogen atoms to form ammonia (NH₃). The nitrogenase reaction additionally produces molecular hydrogen as a side product, which is of special interest for people trying to produce H₂ as an alternative energy source to fossil fuels.

## Anaerobiosis and N₂ Fixation

Nitrogen fixing bacteria have different strategies to reduce oxygen levels, which interfere with nitrogenase function.

### Key Points

* The iron (Fe) found in nitrogenases is very sensitive to oxygen, if there is too much oxygen this will in the end disrupt nitrogenase function.

* Some bacteria produce barriers which protect themselves from oxygen, while others use proteins such as leghemoglobin to bind up oxygen which may interfere with nitrogenases.

* Portions of leghemoglobin are thought to be produced by rhizobia residing in plant nodules, while other parts are produced by the plant, an elegant example of symbiosis.

Central to nitrogen fixation (N₂ to NH₃) are the enzymes that do the actual fixation, these are known as nitrogenases. Due to the oxidation carried out by oxygen, most nitrogenases, which are essential large reduction complexes are irreversibly inhibited by O₂, which degradatively oxidizes the Fe-S cofactors. In essence, O₂ binds to the iron (Fe) found in nitrogenases and blocks their ability to bind to N₂. To protect nitrogenases, there are mechanisms for nitrogen fixers to protect nitrogenase from oxygen in vivo. One known exception is the nitrogenase of Streptomyces thermoautotrophicus, which is unaffected by the presence of oxygen. This is complicated by the fact the bacteria still need the presence of oxygen for proper respiration.

Some microbes have a proteoglycan rich extra cellular matrix which traps a layer of water, often referred to as a **slime layer**. This **slime layer acts as a barrier for oxygen**. The ability of some nitrogen fixers such as azotobacteraceae to employ an oxygen-amendable nitrogenase under aerobic conditions has been attributed to a high metabolic rate, allowing oxygen reduction at the cell membrane.
<figure><img src="image_4.png" /></figure>

**Leghemoglobin:** Leghemoglobin, the protein which binds to oxygen, allowing nitrogenases to function in an oxygen free environment. The ribbons represent protein folds, while the conglomerate of spheres are the position of the iron contain heme group which binds the oxygen.

Many rhizobia, nitrogen fixing bacteria, live in a symbiotic relationship with plants known as legumes. They have an interesting strategy to deal with O₂. In plants infected with Rhizobium, (legumes such as alfalfa or soybeans), the presence of oxygen in the root nodules would reduce the activity of the oxygen-sensitive nitrogenase. In these situations, the roots of such plants produce a protein known as leghemoglobin (also leghaemoglobin or legoglobin). Leghemoglobin buffers the concentration of free oxygen in the cytoplasm of infected plant cells to ensure the proper function of root nodules. Leghemoglobin is a nitrogen or oxygen carrier; naturally occurring oxygen and nitrogen interact similarly with this protein. Leghemoglobin buffers the concentration of free oxygen in the cytoplasm of infected plant cells to ensure the proper function of root nodules. It has close chemical and structural similarities to hemoglobin, and, like hemoglobin, is red in colour. Leghemoglobin has a high affinity for oxygen, about ten times higher than of human hemoglobin. This allows an oxygen concentration that is low enough to allow nitrogenase to function but not so high as to bind all the O₂ in the bacteria, providing the bacteria with oxygen for respiration.

Leghemoglobin is produced by legumes in response to the roots being infected by rhizobia, as part of the symbiotic interaction between the plant and this nitrogen-fixing bacterium. Interestingly, it is widely believed that leghemoglobin is the product of both the plant and the bacterium in which a protein precursor is produced by the plant and the heme (an iron atom bound in a porphyrin ring, which binds O₂) is produced by the bacterium. The
protein and heme come together to function, allowing the bacteria to fix-nitrogen, giving the plant usable nitrogen and thus the plant provides the rhizobia a home.

**Genetics and Regulation of N₂ Fixation**

Through control of gene expression, nitrogen fixing bacteria can turn on and off the proteins needed for nitrogen fixation.

**Key Points**

◇ Nitrogen fixation takes a great deal of energy. If the conditions are not favorable for nitrogen fixation or there is enough ammonia around, nitrogen-fixing bacteria turn off the production of proteins needed for nitrogen fixation.

◇ Nitrogen fixing protein production is regulated by the nif regulon.

◇ The nif regulon contains factors which both turn on and off the production of proteins needed for nitrogen-fixation.

**Key Terms**

✓ **regulon**: A group of genes that is regulated by the same regulatory molecule. The genes of a regulon share a common regulatory element binding site or promoter. The genes comprising a regulon may be located non-contiguously in the genome.

✓ **operon**: A unit of genetic material that functions in a coordinated manner by means of an operator, a promoter, and structural genes that are transcribed together.
NIF REGULON

<figure><img src="image_5.png" /></figure>

**Nif Regulon:** This is a schematic representing many of the proteins in the nif regulon and where they act in the pathway needed for nitrogen fixation.

The fixation of atmospheric nitrogen ($N_2$) is a very energy intensive endeavor. If there is no need for $N_2$ fixation, the production of proteins needed for fixation are tightly controlled. The nif genes are responsible for the coding of proteins related and associated with the fixation of atmospheric nitrogen into a form of nitrogen available to plants. These genes are found in nitrogen fixing bacteria and cyanobacteria. The nif genes are found in both free living nitrogen fixing bacteria and in symbiotic bacteria in various plants.

The nif genes are genes encoding enzymes involved in the fixation of atmospheric nitrogen. The primary enzyme encoded by the nif genes is the nitrogenase complex which is in charge of converting atmospheric nitrogen to other nitrogen forms such as ammonia, which plants can use for various purposes. Besides the nitrogenase enzyme, the nif genes also encode a number of regulatory proteins involved in nitrogen fixation. The expression of the nif genes is induced as a response to low concentrations of fixed nitrogen and oxygen
concentrations (the low oxygen concentrations are actively maintained in the root environment). Nitrogen fixation is regulated by nif regulon, which is a set of seven operons which includes 17 nif genes. Nif genes have both positive and negative regulators. Some of nif genes are: Nif A, D, L, K, F, H, S, U, Y, W, Z.

Activation of nif genes transcription is done by the nitrogen sensitive NifA protein. When there isn't enough fixed nitrogen factor available for the plant's use, NtrC, which is a RNA polymerase, triggers NifA's expression. NifA then activates the rest of the transcription for the nif genes. If there is a sufficient amount of reduced nitrogen or oxygen is present, another protein is activated, NifL. In turn, NifL inhibits NifA activity, which results in the inhibition of nitrogenase formation. NifL is then regulated by other proteins that are sensors for the levels of O₂ and ammonium in the surrounding environment.

The nif genes can be found on bacteria's chromosomes, but many times they are found on bacteria's plasmids with other genes related to nitrogen fixation, such as the genes needed for the bacteria to communicate with the plant host.

## Nitrogen in the Biosphere

* Nitrogen is required in large amounts as an essential component of proteins, nucleic acids, and other cellular constituents.

* Nitrogen is the most abundant element in our atmosphere - nearly 79% in the form of N₂ gas.

* In order for nitrogen to be used for growth it must be "fixed" (combined) in the form of ammonium (NH₄⁺) or nitrate (NO₃⁻) ions.

* Plants are able to use nitrogen in the form of nitrate (NO₃⁻) or ammonia (NH₃⁺), but these compounds are present in limited supply in the soil.

<figure><img src="image_6.png" /></figure>
# Nitrogen Fixation

* The growth of all organisms depend on the availability of Nitrogen (e.g. amino acids)

* Nitrogen in the form of Dinitrogen ($N_2$) makes up 80% of the air we breathe but is essentially inert due to the triple bond ($N=N$)

* In order for nitrogen to be used for growth it must be "fixed" (combined) in the form of ammonium ($NH_4$) or nitrate ($NO_3$) ions.

## Nitrogen Fixation

### Nitrogen Fixation

#### Enzymatic reduction of atmospheric Nitrogen

$$ \underset{\text{(Dinitrogen)}}{\mathbf{N_2}} \xrightarrow[2\text{e}]{2\text{H}^+} \underset{\text{(Di-imide)}}{\mathbf{HN} = \mathbf{NH}} \xrightarrow[2\text{e}]{2\text{H}^+} \underset{\text{(Hydrazine)}}{\mathbf{H_2N-NH_2}} \xrightarrow[2\text{e}]{2\text{H}^+} \underset{\text{(Ammonia)}}{\mathbf{2NH_3}} \\ \boxed{[6\text{e} + 6\text{H}^+ + \mathbf{N_2} \rightarrow 2\text{NH_3}]} $$

#### Energy intensive process :

$$ \mathbf{N_2} + \mathbf{8H}^+ + \mathbf{8e}^- + \mathbf{16\text{ATP}} = \mathbf{2NH_3} + \mathbf{H_2} + \mathbf{16\text{ADP}} + \mathbf{16\text{Pi}} $$

* ☐ Performed only by selected bacteria and actinomycetes

* ☐ Performed in nitrogen fixing crops (eg: soybeans)
# Nitrification

Two step reactions that occur together :

$$1^{\text{rst}} \text{ step: catalyzed by } \text{Nitrosomonas (Slow RK}^{\text{n}}\text{)} \\ 2\text{NH}_{4}^{+} + 3\text{O}_{2} \rightarrow 2\text{NO}_{2}^{-} + 2\text{H}_{2}\text{O} + 4\text{H}^{+}$$

$$2^{\text{nd}} \text{ step: catalyzed by } \text{Nitrobacter (Fast RK}^{\text{n}}\text{)} \\ 2\text{NO}_{2}^{-} + \text{O}_{2} \rightarrow 2\text{NO}_{3}^{-}$$

# Nitrogen Fixation Process

## Energetics

* N≡N

* Haber-Bosch (100-200 atm, 400-500°C, 8,000 kcal kg⁻¹N)

* Nitrogenase (4,000 kcal kg⁻¹N)

### Properties of dinitrogen which makes it inert

* Dinitrogen - two N atoms connected by triple bond

* Breaking the N≡N bond is difficult - high dissociation energy of 942 kJ mol⁻¹

* Breaking first π bond requires 540 kJ mol⁻¹

<figure><img src="image_7.png" /></figure>
Other important properties

<table><thead><tr><th>Gas</th><th>Nitrogen</th><th>Carbon</th><th>Oxygen</th><th>Argon</th></tr></thead><tbody><tr><th scope="row">Property</th><td></td><td></td><td></td><td></td></tr><tr><th scope="row">Ionization potential (eV)</th><td>14.3</td><td>11.256</td><td>13.614</td><td>15.755</td></tr><tr><th scope="row">Electron affinity (eV)</th><td>0.073</td><td>1.595</td><td>1.461</td><td>0</td></tr><tr><th scope="row">Solubility in water (mole/cm³)</th><td>0.083</td><td>Insoluble</td><td>0.153</td><td>0.140</td></tr></tbody></table>

☐ High ionization potential and low electron affinity - difficult to reduce and oxidize

☐ Solubility very less - reactions in solution phase - difficult

Biological fixation of dinitrogen

☐ Enzyme nitrogenase

☐ Present in soil bacteria, root nodules and algae

☐ Two decades of research - mechanism not established

☐ Enzyme contains Mo and Fe

☐ Proposed mechanism - complexation of N₂ to metal ions

☐ Reduces bond strength - breaking 1<sup>st</sup>π bond easier

Limitations with biological route:

☐ Nitrogenase - sensitive to O₂ - requires O₂ free environment

☐ Sensitive to environmental conditions - temperature, pH

☐ Cannot be used for large scale N₂ fixation

Different types of associations:

<table><thead><tr><th>Types of association</th><th>Microorganisms</th><th>Host plants</th></tr></thead><tbody><tr><td rowspan="3">Symbiotic</td><td>Bacteria (ex. Rhizobium)<sup>a</sup></td><td>Legumes</td></tr><tr><td>Actinomycetes (ex. Frankia)</td><td>Actinorhiza</td></tr><tr><td>Cyanobacteria (ex. Anabaena azollaea)</td><td>Fern</td></tr><tr><td>Non symbiotic</td><td>Bacteria (ex. Azotobacter, Azospirillum)</td><td>Cereals</td></tr><tr><td>Free living systems</td><td>Bacteria (ex. Thiobacillus, Clostridium)</td><td></td></tr></tbody></table>
aSix main genus of Rhizobia:

1. Allorhizobium (fast growing)

2. Azorhizobium (fast growing)

3. Bradyrhizobium (slow growing)

4. Mesorhizobium (intermediate growing)

5. Rhizobium (fast growing)

6. Sinorhizobium (fast growing)

Key Terms

➢ **Fixation**: The act of uniting chemically with a solid substance or in a solid form; reduction to a non-volatile condition.

➢ **Cofactor**: A substance, especially a coenzyme or a metal, that must be present for an enzyme to function.

➢ **Heterometal**: Describing a complex containing two or more different metals

➢ **Symbiosis**: A close, prolonged association between two or more organisms of different species, regardless of benefit to the members

➢ **Sulfide**: Any compound of sulfur and a metal or other electropositive element or group.

➢ **Reduction**: A reaction in which **electrons are gained** and valence is reduced; often by the **removal of oxygen** or the **addition of hydrogen**.

➢ **Enthalpy**: In thermodynamics, a **measure of the heat content of a chemical** or physical system.

➢ **Oxidation**: A reaction in which the atoms of an element **lose electrons** and the valence of the element increases.

➢ **Proteoglycan**: Any of many glycoproteins that have hetero-polysaccharide side chains.

Genetics of Nitrogenase

<table><thead><tr><td>Gene</td><td>Properties and function</td></tr></thead><tbody><tr><td>nifH</td><td>Dinitrogenase reductase</td></tr><tr><td>nifDK</td><td>Dinitrogenase</td></tr><tr><td>nifA</td><td>Regulatory, activator of most nif and fix genes</td></tr><tr><td>nifB</td><td>FeMo cofactor biosynthesis</td></tr><tr><td>nifEN</td><td>FeMo cofactor biosynthesis</td></tr><tr><td>nifS</td><td>Unknown</td></tr></tbody></table>
<table><tr><td>fixABCX</td><td>Electron transfer</td></tr><tr><td>fixK</td><td>Regulatory</td></tr><tr><td>fixLJ</td><td>Regulatory, two-component sensor/effector</td></tr><tr><td>fixNOQP</td><td>Electron transfer</td></tr><tr><td>fixGHIS</td><td>Transmembrane complex</td></tr></table>

## Nitrogen fixing organisms

* Free living aerobic bacteria

* Azotobacter

* Beijerinckia

* Klebsiella

* Cyanobacteria
    (lichens)

* Free living anaerobic bacteria

* Clostridium

* Desulfovibrio

* Purple sulphur bacteria

* Purple non-sulphur
    bacteria

* Green sulphur bacteria

* Free living associative bacteria

* Azospirillum

* Symbionts

* Rhizobium
    (legumes)

* Frankia (alden trees)

## Free-living N₂ Fixation

### Energy

* 20-120 g C used to fix 1 g N

### Combined Nitrogen

* *nif* genes tightly regulated

* Inhibited at low NH₄⁺ and NO₃⁻ (1 µg g⁻¹ soil, 300 µM)

### Oxygen

* ✓ Avoidance (anaerobes)

* ✓ Microaerophilic Respiratory protection

* ✓ Specialized cells (*heterocysts*, vesicles)

* ✓ Spatial/temporal separation

* ✓ Conformational protection
## Associative N₂ Fixation

* Phyllosphere or rhizosphere (tropical grasses)

* Azospirillum, Acetobacter

* 1 to 10% of rhizosphere population

* Some establish within root

* Same energy and oxygen limitations as free-living

* Acetobacter diazotrophicus lives in internal tissue of sugarcane, grows in 30% sucrose, can reach populations of $10^6$ to $10^7$ cells g⁻¹ tissue, and fix 100 to 150 kg N ha⁻¹ y⁻¹

## Phototrophic N₂-fixing Associations

* Lichens-cyanobacteria and fungi

* Mosses and liverworts-some have associated cyanobacteria

* Azolla-Anabaena (Nostoc)-cyanobacteria in stem of water fern

* Gunnera-Nostoc-cyanobacteria in stem nodule of dicot

* Cycas-Nostoc-cyanobacteria in roots of gymnosperm

## Formation of a Root Nodule

<figure><img src="image_8.png" /></figure>

### Infection Process:

* Attachment

* Root hair curling
* Localized cell wall degradation

* Infection thread

* Cortical cell differentiation

* Rhizobia released into cytoplasm

* Bacterioid differentiation (symbiosome formation)

* Induction of nodulins

## Role of Root Exudates:

### General

* Amino sugars, sugars

### Specific

* Flavones (luteolin), isoflavones (genistein), flavanones, chalcones

* Inducers/repressors of *nod* genes

* Vary by plant species

* Responsiveness varies by rhizobia species

<figure><img src="image_9.png" /><figcaption>Luteolin</figcaption></figure>

<figure><img src="image_10.png" /><figcaption>Genistein</figcaption></figure>

## Nodule Metabolism:

<figure><img src="image_11.png" /></figure>

### Oxygen metabolism

* ✓ Variable diffusion barrier

* ✓ Leghemoglobin

<figure><img src="image_12.png" /></figure>

### Nitrogen metabolism

* NH₃ diffuses to cytosol

* Assimilation by GOGAT

* Conversion to organic-N for transport

<figure><img src="image_13.png" /></figure>

### Carbon metabolism

* ✓ Sucrose converted to dicarboxylic acids

* ✓ Functioning TCA in bacteroids

* ✓ C stored in nodules as starch

<figure><img src="image_14.png" /><figcaption>ALLANTOIN (C:N=1)</figcaption></figure>

<figure><img src="image_15.png" /><figcaption>ASPARAGINE (C:N=2)</figcaption></figure>

<figure><img src="image_16.png" /><figcaption>ALLANTOIC ACID (C:N=1)</figcaption></figure>

<figure><img src="image_17.png" /><figcaption>GLUTAMINE (C:N=2·5)</figcaption></figure>

<figure><img src="image_18.png" /><figcaption>CITRULLINE (C:N=2)</figcaption></figure>

<figure><img src="image_19.png" /><figcaption>Y METHYLENE GLUTAMINE (C:N=3)</figcaption></figure>

## Nitrogen Fixation

* All nitrogen fixing bacteria use highly conserved enzyme complex called **Nitrogenase**
* Nitrogenase is composed of two subunits: an **iron-sulfur protein** and a **molybdenum-iron-sulfur protein**

* Aerobic organisms face special challenges to nitrogen fixation because nitrogenase is inactivated when oxygen reacts with the iron component of the proteins.

<figure><img src="image_20.png" /></figure>

<figure><img src="image_21.png" /></figure>

<figure><img src="image_22.png" /></figure>

<figure><img src="image_23.png" /></figure>

Nitrogenase

<figure><img src="image_24.png" /></figure>
<figure><img src="image_25.png" /><figcaption>Nitrogenase proteins and cofactors. (A) Shown is one half of the nitrogenase complex, with one αβ pair of the MoFe protein α₂β₂ tetramer and one bound Fe protein dimer. The location of bound nucleotides and the three metal clusters are also shown.</figcaption></figure>

Barney BM, et al., Dalton Trans. 2006, 2277-84.

<figure><img src="image_26.png" /></figure>