Biochemistry of Nitrogen Fixation (With Diagram)

The below mentioned article provides an Overview on Biochemistry of Nitrogen Fixation. After reading this article you will learn about: 1. Enzymology 2. Substrate 3. Non-Symbiotic Fixation 4. Symbolic N₂– Fixation 5. Exchange of Metabolites between Bacteroids and Host Cells.

Contents:

1. Enzymology
2. Substrate
3. Non-Symbiotic Fixation
4. Symbolic N2– fixation
5. Exchange of Metabolites between Bacteroids and Host Cells

1. Enzymology:

The biological reduction of di-nitrogen to ammonia is catalysed by nitrogenase enzyme complex, consisting of two enzyme components — di-nitrogenase (MoFe-protein) and di-nitrogenase reductase (Fe-protein). The former is a 240-kDa heterotetramer that binds N₂ and holds it while it is being reduced and the latter is a 64-kDa homodimer that provides di-nitrogenase with high-energy electrons.

The subunits of the Fe-protein are identical and the dimer contains one Fe₄S₄ cluster. It is extremely sensitive to oxygen (half-life in air is ~ 30 sec). The midpoint reduction potential of the reaction catalysed by Fe-protein is about – 400 mV when the enzyme is binding ATP.

Di-nitrogenase (MoFe-protein) is an α₂β₂ heterotetramer containing two pairs of unusual metal clusters as integral components of the enzyme. These are called P-clusters each having 8Fe-7S iron- sulfur complexes. In its reduced state (P^N), a P-cluster resembles two distorted 4Fe-4S cubes that share one sulfur at the corner. In the oxidized state (P^ox), one of the cubes opens, breaking two of the bonds with this corner sulfur (Fig. 10.5).

The P-clusters are the catalytic centers. In addition a second pair of metal clusters is held in di-nitrogenase by only a cysteinyl sulfur and a histidine nitrogen. This is called the MoFe cofactor (MoFeCo), which is a large redox center made up of Fe₄S₃ and Fe₃MoS₃. Another constituent of the cofactor is homocitrate, which is linked via oxygen atoms of the hydroxyl group to molybdenum.

The enzyme hydrogenase was described for the first time by Gest and Kamen in 1949 in Rhodospirillum rubrum. The enzyme catalyses the activation of molecular hydrogen.

If the substrate (nitrogen) is not available the electrons combine with protons to release H₂ gas. ATP-dependent H₂ evolution is a property of the nitrogenase complex and is quite distinct from ATP-independent evolution of H₂ by hydrogenase which is found in nitrogen-fixing cells. All nitrogen- fixing organisms possess hydrogenase, but not all organisms which have hydrogenase fix nitrogen.

2. Substrate:

In addition to nitrogen there are many other substrates which are reduced by the enzyme nitrogenase like N₂O, N₃, HCN, C₂H₂, etc. These are the non-physiological substrates. Acetylene is reduced to ethylene by the enzyme as it is very similar to N₂ in its properties. H₂ is a competitive inhibitor of N₂-reduction in cell free extracts as well as in intact cells.

In presence of sufficient concentrations of acetylene only this is reduced to ethylene:

HC = CH + 2e^– + 2H⁺→H₂C = H₂C

This reaction is used to measure the activity of di-nitrogenase. It is called the acetylene reduction test.

---

ADVERTISEMENTS:

3. Non-Symbiotic Nitrogen (N₂)-Fixation:

In 1960, J. E. Carnahan and his associates first reported the reduction of gaseous nitrogen to ammonia by the cell-free preparations from the bacterium, Clostridium pastcurianum (Fig. 10.6). They observed that a large amount of pyruvic acid was required by the extract for the fixation. The pyruvic acid through phosphoroclastic dissimilation was degraded to acetyl phosphate, CO₂ and hydrogen.

The extract was separated into two components:

(i) The hydrogen-donating component, through which the electrons flow from the pyruvic acid via ferredoxin to the second component,

(ii) The nitrogen-reducing component that reduces the molecular nitrogen to ammonia. Pyruvic acid given to the extract as the source of ATP and a low redox potential reductant, ferredoxin, a brown iron-sulfur protein is required for the enzyme system. Under in vitro reaction, anaerobic conditions are absolutely required for the steady enzyme activity.

(a) Phosphoroclastic Dissimilation:

Pyruvic acid through a specific reaction called phosphoroclastic dissimilation provides reducing power and ATP. In this reaction pyruvic acid is degraded to acetyl phosphate, CO₂ and H₂.

This phosphoroclastic reaction has also been reported in photosynthetic bacteria and cyanobacteria. This work of Carnahan and his associates thus opened the avenue for a detailed analysis of this important series of reactions.

Now a generalization can be made regarding the essential reaction components as follows:

(a) An electron-donating system,

(b) An electron-carrying system,

(c) An electron acceptor (i.e., N₂ gas),

(d) ATP together with a divalent cation Mg^{2 +}, and

(e) Nitrogenase system (Fe-protein and MoFe-protein).

(b) Electron Donors:

Pyruvic acid and NADPH are the electron donors in most of the cases. Hydrogen gas can also serve as the electron donor. Other sources of electrons are the photolysed water, the assimilated carbon and reduced sulphur compounds.

It is also evident that the electron-donating systems could be PS-I, the glycolysis and Krebs cycle and the pentose phosphate pathway. Still it is uncertain whether more than one type of electron-donating system can operate simultaneously.

(c) Mg-ATP:

Mg-ATP is absolutely required as the energy source for nitrogen fixation as the process is highly energy demanding. The Fe-protein, but not the MoFe-protein, binds with Mg-ATP. The binding of Mg-ATP to the Fe-protein gives a conformational change of the protein so that it becomes more sensitive to O₂.

The utilized product Mg-ADP, strongly inhibits nitrogenase catalysis. That is why the growing N₂-fixing cells, need ATP: ADP ratio between 2: 1 and 3:1. Other cations like Mn²⁺, Fe²⁺, Co²⁺ and Ni²⁺ are less effective and do not support the reaction.

(d) Electron Carriers:

In 1962 Mortensen and Carnahan working at the Du Pont Laboratories discovered an important electron carrying iron-sulphur protein, ferredoxin, from the bacterium Clostridium pasteurianum. The protein has an extremely low redox potential at a slightly alkaline pH.

Its molecular weight ranges from 10,000 to 40,000. 2-4 iron-sulphur atoms are present per molecule. It functions in nitrogen fixation by connecting the hydrogen-donating and the nitrogen-activating systems.

A related but distinct electron carrier for nitrogen fixation is flavodoxin, which is produced in place of ferredoxin when the bacterium grows on a limited supply of iron. Flavodoxin has been subsequently detected in several organisms. Both ferredoxin and flavodoxin may operate simultaneously in some cases. However, they have been shown to donate electrons in vitro to nitrogenase.

(e) Six-electron Requirement:

In nature nitrogen may exist in either a highly oxidized form (NO₃ ^–) or in a highly reduced state (NH₃), according to the oxidation number as shown below:

Recently, a proposal with an eight-electron requirement has been suggested, as H₂ evolution takes place during N₂-fixation, but support for the enzyme stoichiometry is lacking.

The following four equations indicate the direction of election flow (Fig. 10.7):

The reaction mechanism is same in both free-living as well as symbiotic organisms.

(f) Intermediates in N₂-fixation:

By the application of ¹⁵N₂, the role of ammonia (ammonia hypothesis) in N₂-fixation was firmly established. However, an alternative hypothesis, i.e., hydroxylamine hypothesis was offered before the acceptance of ammonia hypothesis.

The former hypothesis holds that N₂ is reduced to NH₂OH which combines with keto acid to form oximes which in turn are reduced to amino acids. There are many evidences supporting the ammonia hypothesis.

The formation of ammonia can be demonstrated directly. Cultures of Clostridium pasteurianum excrete NH+ when grown on a medium that supplies inadequate a-ketoglutaric acid to serve as an acceptor for the NH⁺. If the cultures are supplied with ¹⁵N₂ for a short period, the highest ¹⁵N₂ accumulation was found in NH₄⁺.

4. Symbolic Nitrogen (N₂)–Fixation:

Research on free-living microbes has essentially contributed to N₂-fixation concepts. However, symbiotic N₂-fixation involving both legumes and Rhizobia is unique and ecologically they contribute much to biological N₂– fixation. The mechanism of reaction is the same as that of the free-living systems but varies by some additional steps (Fig. 10.9).

Only three types of N₂-fixing bacteria have evolved beneficial symbiotic association with higher plants — (a) Rhizobium sp. forms root nodules on leguminous plants, (b) Frankia (actinomycete-like soil organism) forms root nodules on Alnus, Purshia, etc., and (c) the heterocystous blue-green algae, establishing symbiotic relationship with a variety of hosts. Of these, the rhizobial symbiosis has been studied in greater detail.

The principal purpose of the symbiont in the symbiotic N₂^–fixation is the formation of NH₄⁺ for the host. The bacteroids lack a rigid cell wall and are osmotically labile. They are known to metabolize various carbon compounds supplied by the plant cell.

The major carbon substrates found in nodules are carbohydrates, polyols (carbohydrates with alcohol side groups) and organic acids. Rhizobia may change their substrate requirement when they differentiate from free-living into bacteroid forms.

The specific permeases present in the bacteroid membrane control the transport of organic compounds into the bacteroid cytoplasm. The transported carbon compounds are metabolized via the TCA cycle coupled to the energy-yielding respiratory chain capable of functioning under low O₂ concentrations.

Higher O₂ concentrations inactivate the O₂ sensitive nitrogenase activity. Leg hemoglobin functions as an oxygen carrier maintaining a sufficient and constant O₂ concentration near the bacteroid membrane.

Two additional membrane-bound systems may play an important role in energy metabolism — (a) the H₂ uptake system and (b) the energy-linked nitrate reductase system. Most H₂-utilizing bacteria induce a membrane-bound H₂ uptake system, presumably coupled to the major respiratory chain.

Carter et al. (1 980) purified a ferredoxin from Rhizobium japonicum and showed that it could reduce Fe-protein. However, it is still not clear as to how ferredoxin is reduced.

One suggestion is that an intact bacterial membrane could help in the flow of electrons to ferredoxin if either:

(i) the membrane potential is sufficiently large

(ii) there is a proton motive force sufficient to drive reverse electron flow.

A combination of both is also possible.

The nitrogenase complex, consisting of di-nitrogenase reductase (Fe-protein) and di-nitrogenase (Mo-Fe-protein) catalyses the nitrogen fixation. The enzyme remains in the cytoplasm of the bacteroids From NADH, formed in the TCA cycle, electrons are transferred via a soluble electron carrier, ferredoxin to di-nitrogenase reductase.

The latter is a single electron carrier. It is a homodimer, both subunits of which form a Fe₄-S₄ cluster and contain two ATP binding sites. After the reduction of di-nitrogenase reductase, Mg-ATP molecules bind to it, bringing about a conformational change of the protein as a result the redox potential of the Fe₄-S₄ cluster is raised from – 0.25 to – 0.40 V.

Following the transfer of an electron to di-nitrogenase the bound ATP molecules are hydrolysed to ADP and P and then released from the enzyme surface, restoring the low redox potential of the enzyme which becomes ready again to take up another electron from ferredoxin. Thus, two molecules of ATP are consumed per electron transferred from NADH to di-nitrogenase via di-nitrogenase reductase.

5. Exchange of Metabolites between Bacteroids and Host Cells:

Malate, formed from sucrose, is the principal translocate provided by the host cells to the bacteroids. So, the supply of sucrose and its metabolism is very crucial for the host-bacteroid cooperation. Sucrose is delivered to the host cells adjacent to the bacteroids through sieve tubes of the phloem supply. Sucrose is synthesized by sucrose synthase and degraded through glycolysis.

The glycolytic intermediate phosphoenolpyruvate (PEP) is carboxylated to oxaloacetate (OAA) which is then reduced to malate. PEP carboxylase activity is very high in the nodular host cells. Malate is then transported into the bacteroids through the malate trans locator built in the symbiosome membrane.

Malate inside the bacteroids is oxidized through TCA cycle and the derived reducing power is utilized in N₂-fixation. The fixation product, NH⁺₄ is delivered via a specific channel on the pen-bacteroid membrane to the host cell. The NH; is mainly metabolized to glutamine and asparagine that are then transported as ureides (allantoin and allantoic acid), which have a high N/C ratio.

(a) Leg-Hemoglobin:

Kubo in 1939 correlated the pink colouration of the legume nodules with the pigment leg-hemoglobin. This is very much similar in properties to hemoglobin of red blood corpuscles Leg-hemoglobin is a symbiotic compound, as the two parts of the molecule, an Apo protein and a heme, being coded for by the DNA of both the host plant and Rhizobium, respectively.

Previously it was thought that these two components form an active complex, extracellularly to both partners but within the pen-bacteroid membrane envelope. However, recent work shows that most of the hemoglobin is located in the host cell cytoplasm but a physiologically significant amount bathes the bacteroids within their envelops.

Now there is an overwhelming evidence that leg-hemoglobin acts as an O₂ carrier. The nodules need sufficient oxygen to reach the bacteroids for ATP synthesis without causing inactivation of nitrogenase, that is, a high flux of O₂ at a low concentration.

The leg-hemoglobin performs this function by its O₂ binding properties. Oxygen at a low concentration diffused into the nodule is consumed by the bacteroid membrane ETS. Due to very high affinity of the bacteroid cytochrome oxidase complex for O_2, respiration is still possible with such concentration (10^-9 mole 1^-1).

A total of 16 ATPs is required for the fixation of one molecule of N₂. Two molecules of ATP per NADH re-oxidized might be formed through bacterial ETS. Thus, about four molecules of O₂ have to be consumed to produce 16 molecules of ATP. Oxygen demand may be further increased due to hydrogenase action (O₂/N₂> 4). But the O₂/N₂ ratio in air is about 0.25.

So, it needs a steady supply of oxygen to the bacterial respiratory chain. This is done by leg-hemoglobin present in the peribacteroid membrane. Since the bacterial respiratory chain is located in the membrane and di-nitrogenase in the interior of the bacteroids, O₂ is kept in a safe distance from di-nitrogenase to avoid inactivation.

Leg-hemoglobin is only the most obvious molecule in the nodule. Now genes are known to code for 18-20 proteins termed nodulins which are only produced in the host cells of the effective nodules (Anger and Verma, 1981).

These nodulins includes the enzymes of carbohydrate degradation, the TCA cycle, glutamine and asparagine synthesis, ureide synthesis, malate trans-locator on the peribacteroid membrane and leg-hemoglobin. Nodulin genes may be early or late. The early genes are involved in the process of infection and formation of nodule. The late genes are expressed only after the formation of the nodules.

(b) Energetics:

Recent in vitro studies have reported a minimal requirement of 12-15 moles ATP per mole of N, reduced, but the values vary with experimental conditions. Estimates of the in vivo energy requirement for various organisms vary from 4-5 to 29 ATP/N₂ according to the following table (Table 10.1).

So, the in vivo experiments indicate that N₂-fixation requires a very large input of metabolf energy. The Davis group has suggested finally a total of 35-40 ATP/N, for the appropriate reaction catalysed by nitrogenase.

(c) nif Gene Organization:

A large number of recombination studies and transduction analyses have shown that the region of the nif gene operon is 15-20 gene-length in size. Valentine’s group in 1978 reported that most nif genes are clustered near the histidine operon.

Recently, Haselkorn’s group has determined the nucleotide sequence of the structural gene coding for Fe-protein of N₂ase from Anabaena. In Klebsiella pneumonia, at least 20 genes, linked and arranged in about seven transcription units are involved in N₂– fixation.

The minimum size of the entire nif gene cluster has been found to be about 24 kilobase (1 kilobase = 1000 nucleotide pairs). This entire region has been sequenced.

(d) Nitrogen Fixation in Cyanobacteria:

Certain cyanobacteria have nitrogen-fixing ability. Among free-living diazotrophs, they have a distinct advantage in that, as photoautotrophs, the energy and reducing power required for N₂– fixation is supplied by light. The diazotrophic cyanobacteria possess an oxygen evolving photosystem- II.

So, these two processes, N₂-fixation and photosynthesis, are metabolically incompatible in the same cell at the same time. Being prokaryotes the cyanobacteria do not possess any organelle apart from the thylakoids (chromatophores). They are unlikely, therefore, to effect an intracellular compartmentalization of the two processes.

Nevertheless, cyanobacteria have evolved devices for this paradox. The present-day unicellular forms separate the two processes in time, while the multicellular, filamentous forms effect a spatial separation of photosynthesis and N₂-fixation. To achieve this, the latter forms produce a specialized, differentiated cell called the heterocyst.