Nitrogen Fixation Mechanisms in Microorganisms | Microbiology

In this article we will discuss about the nitrogen fixation mechanisms in microorganisms.

1. Nitrogenase Types, Structure and Function:

The biological conversion of atmospheric nitrogen to ammonia taken place with the help of an enzyme called Nitrogenase. This enzyme is anaerobic in nature and when it comes in contact with oxygen or air, it becomes inert. The proteinaceous enzyme is made up of 2 subunits mainly called larger MoFe protein (2,20,000 Dalton mol. wt.) and another smaller Fe protein (55000 Dalton mol. wt.).

Postgate, a British microbiologist named them as Kp₁ and Kp₂. As stated it contains iron and molybdenum atoms. They need Mg²⁺ ions for activation and can convert ATP to ADP during functioning.

It is inhibited by ADP and also reduces several other substrates with triple bonded molecules (similar to N = N). The enzyme can reduce hydrogen ions to gaseous hydrogen, even when N₂ is present, and also have an ability to, reduce acetylene to ethylene.

The nitrogenase purified from three species of bacteria showed its following nature as given below. This indicates that nature of enzyme varies in different nitrogen fixing organisms as far as the size of proteins (in both MoFe and Fe components) is concerned.

(i) The Cp Type:

According to Postgate, the properties of the nitrogenase are the Cp type (Clostridium pasteurianum): It has MoFe (Cp₁) and Fe (Cp₂) proteins which have 2,20000 Dalton and 55,000 Dalton molecular weight, respectively. The half-life of enzyme is quite short.

(ii) The Kp Type (Klebsiella pneumoniae):

It has MoFe (Kp₁) and Fe (Kp₂) which have 2,18000 Daltons and 66,700 Dalton molecular weight respectively.

(iii) The Ac Type (Azotobacter chroococcum):

It has MoFe (Ac₁) and Fe (Ac₂) which have 2,27000 and 64,000 Dalton molecular weight respectively.

It is observed that the half-life of enzyme of all the three types of Fe units are much shorter than that of FeMo units.

2. Alternative Nitrogenase:

Professor P. Bishop and his colleagues in USA obtained evidence that Azotobacter vinelandii has a different kind of nitrogenase. The genetic evidences revealed that their normal genes for nitrogenase (nif YKDH) deleted. This nitrogenase was later isolated from A. chroococcum. It consists of two proteins, one large and heteromeric, one smaller very like the regular Fe-protein; both are sensitive to O₂.

The enzyme evolves one molecule of H₂ and reduces acetylene. The larger protein subunit part of enzyme contains vanadium in place of Mo ion of the conventional system in this ‘new’ nitrogenase. V-nitrogenase repressed by Mo suggests that it provides Azotobacter with a physiological ‘back-up’ nitrogen-fixing system for use in case of lack of Mo.

3. Substrates for Nitrogenase:

For enzyme activity a suitable substrate is required so as to bind all the active sites of enzyme to get a product.

The overall reaction in the enzymic reduction of atmospheric nitrogen to ammonia could be postulated as follow:

It is interesting to note that cell free extract of Azotobacter and Clostridium converted nitrogen in the same way as the free-living bacterial cells. This finally led to the initial isolation and purification of the enzyme from C. pasteurianum and A.chroococcum. Further, the enzyme was responsible for the adsorption and reduction of N₂ gas.

Although there are several substrates of nitrogenase but except H₂ most of the substrates are non-physiological substrates because the inhibitors are actually reduced by nitrogenase. Besides this, acetylene (HC=CH) is an important and one of the reliable substrates for measuring the enzyme activity by ‘acetylene-reduction’ test. It is also important to note that most of the substrates have triple bond in their molecules similar to nitrogen (N=N).

Following are the substrates reactive to nitrogenase:

Substrate – Products(s)

N₂ – NH₃

N₂O – H₂O, N₂

N₃ – NH₃, N₂

C₂H₂ – C₂H₄

HCN – CH₄, NH₃, CH₃, NH₂

CH₃CN – C₂H₆, NH₃

In the reduction of acetylene, the product formed is ethylene i.e. HC = CH → H₂C-CH₂ which requires two electrons, whereas reduction of nitrogen to ammonia requires six electrons. Nitrogen is readily reduced in comparison to rest of the substrates.

If there is N₂ fixation, simultaneously H₂ is also produced by some of the nitrogen fixing- microorganisms. N₂ fixation is correlated with each other. Nitrogenase reduced the H⁺ ion and formed H-D in the presence of deuterium. The H-D reaction suggests the involvement of a bound di-imide intermediate in N₂ fixation.

In view of H₂ evolution during nitrogen fixation, the following reaction has been suggested:

N₂ + 8 H⁺ + 8e^– +16 ATP → 2NH₃ + H₂ + 16 ADP + 16 Pi

The energy for nitrogenase reaction comes from the cellular metabolic cycles in the form of ATP. This is met out by photophosphorylation, oxidative phosphorylation or phosphoroclastic dissimilation. In later process, keto-acid is dissimilated to acetyl phosphate, CO₂ and H₂.

Pyruvate functions both as electron donor and as energy source. In the phosphoroclastic reactions, pyruvate forms acetyl phosphate which in the presence of ADP gives rise to ATP. The reductants are the naturally occurring electron carrier proteins called ferredoxin md flavodoxin.

Dithionite (Na₂S₂O₄) and certain dyes such as methyl viologen and benzyl viologen can also serve as artificial extracellular sources of electron donors. This enzyme system catalyzes the transfer of electrons from pyruvate or hydrogen to ferredoxin or flavodoxin.

(a) Ferredoxins or di-nitrogenase:

Ferredoxins are electron carrier, discovered by Mortenson and Caruahan in the year 1962 from C. pasteurianum. It is naturally occurring e^– carrier iron-sulphur (Fe-S) protein (reversible). It has now been isolated from number of cyanobacteria, photosynthetic bacteria and even from higher plants.

The ferredoxins are involved in various physiological processes such as photosynthesis in plants and pyruvate metabolism in anaerobic bacteria. The ferredoxin involved in N₂ fixation contains one cluster of four irons and four sulphur atoms in the molecule. Many similar iron-sulphur clusters are the part of iron atoms of nitrogenase. The whole cluster behaves as oxido-reductive unit.

The electron paramagnetic resonance indicates that the ferredoxins of aerobic nitrogen fixing bacteria such as Azotobacter behave slightly differently from those of anaerobes such as Clostridium pasteurianum. In C. pasteurianum, ferredoxin is the actual protein which reacts with nitrogenase and provides the reducing power for the conversion of N₂ to NH₃.

(b) Flavodoxins or di-nitrogen reductase:

The bacteria grow under limited iron supply i.e. nutritional stress condition and produce flavodoxin. It was also isolated in the beginning from C.pasteurianum. It is interesting that it was found to replace ferredoxin as an electron carrier in a large number of reactions. An electron carrier named ‘azotoflavin’ has been isolated from Azotobacter vinelandii possessing biological activity similar to ferredoxins.

In K.pneumoniae and A.chroococcum, flavodoxins are the primary reductant to nitrogenase. A. chroococcum flavodoxin is blue when half-reduced and colourless when fully reduced. The flavodoxin reduced form is quinone and semi-quinone form.

This form is unusually stable to oxidation by air, which may be why this protein rather than a ferredoxin is particularly suitable to Azotobacter’s aerobic way of life. Flavodoxins do not contain iron-atoms; their oxido-reducible centre is yellow, fluorescent molecule called a ‘flavin’.

Role of Pyruvate and Ferredoxin-Nitrogenase Reaction:

The Fig. 14.6 shows the active site of the enzyme for substrate reduction. This enzyme is believed to be composed of a Mo-Fe di-nuclear site bridged by sulphur having the proper size and electron characteristics to provide Mo-Fe distance of 3.8 Å.

This distance is specific so as to accomodate various nitrogenase substrates including nitrogen and to exclude others. The first reaction in nitrogen reduction is the formation of a linear complex of nitrogen with the Fe atom of nitrogenase.

This is followed by transfer of electrons from Mo which is the end point of the electron activating system resulting in the formation of di-imide which is stabilized by hydrogen bonding from the protein as well as the metal nitrogen bonds.

Successive addition of electrons produces hydrazine followed by cleavage of N—N bond to fill two molecules of NH₃. The increase in the N—N bond length during reduction is accompanied by compensating changes in the MNN angles so that Mo—Fe distance remains constant.

4. Electron Proteins:

MoFe (kp₁) proteins plays a key role in nitrogen fixation (substrate binding and reduction) with Fe protein (kp₂) that assists in transfer of electron from flavodoxin to the bigger subunits and ATP consumption. Neither of the two proteins subunits can function independently (Fig. 14.7).

Fe proteins has four Fe centres and equal number of sulphur, whereas the number of Fe centres in MoFe protein varies. It is 22-24 in C.pasturianum. An equivalent number of inorganic sulphur is also present. In contrast to Fe protein, MoFe protein possesses two additional Mo atoms. Mo is suggested to play a vital role in H₂ evolution which accompanies N₂ fixation.

N₂ reduction process starts with the transfer of single electron (2 ATP molecules) from flavodoxin to smaller Fe protein subunit Kp₂. At this stage two ATP molecules combine with two ions to form complex, Mg ATP which attaches to Fe protein and energizes it to transfer electron from the iron atoms in Fe protein to MoFe protein, prior reaching the bound substrate.

The MoFe protein becomes unstable and to counter balance this electron a H⁺ ion formed by dissociation of H₂O comes to attach at Mo atom of MoFe protein. The electron flow from flavodoxin to MoFe protein via Fe protein is a repeated process. Each time an electron is transferred; there is a consumption of two molecules of ATP.

The second electron is again balanced by another H⁺ ion. When the third electron is transferred to MoFe protein, the H⁺ ions are displaced by nitrogen leading to the evolution of one molecule of hydrogen. This third electron again balances by attachment of one H⁺ ion to nitrogen i.e. HN = NH.

This process of electron transfer is continued till net 8 electrons transfer thereby reducing N, to NH₃. The series of reactions involved by electron transfer is depicted in Fig. 14.7.

H⁺ ———— Counter balance 1st e^– (electron)

H⁺ ————- Counter balance 2nd e^– (electron)

H⁺ + H⁺ → H₂ evolved

H+ …. N=N ….. H⁺ ← N=N

(3rd e^–) (4th e^–)

H+ … HN.—— .NH . H⁺

(5th e^–) (6th e^–)

H⁺ ….. H₂N.———- .NH₂ …….. H+

(7th e^–) (8th e^–)

NH₃ + NH₃

i.e. 2NH₃

Thus, net 8 e^– and 8H⁺ are involved in reducing N₂ to two molecules of ammonia. One electron utilizes two moles of ATP. Hence, net 16 moles of ATP are used. The H₂ evolution is mediated by transfer of net two electrons, thereby leading to the loss of four ATP molecules. It is obvious that the process of nitrogen reduction consumes 12 ATP and the complete process of biological nitrogen fixation is still more expensive.

The enzyme reaction should be written formally as:

N₂ + 8H⁺ + 16 ATP + 8 e^– → 2 NH₃ + H₂ + 16 ADP

Nitrogen fixation is a reductive process where N₂ is reduced to give NH₃, an inorganic product. Such studies have been confirmed by autoradiography (use of ¹⁵N₂). On the other hand, the first organic product formed is glutamic acid.

On the basis of oxidation number, following scale is proposed:

On the above scale, NO3 is highly oxidized and ammonia is highly reduced. Therefore, during nitrogen fixation, a continuous reduction ranges from oxidation number zero to – 3.

5. Hydrogen Evolution:

For every N₂ molecule fixed, one molecule of H₂ is evolved. This process is expensive to nitrogen fixation due to involvement of 16 ATP molecules, whereas similar fixation can be held by using 12 ATP molecules.

It means four molecules are just wasting. To overcome this process, the enzyme responsible for normal hydrogen evolution (i.e. that formed in the normal metabolism of these bacteria, not via nitrogenase) is called hydrogenase, and it can catalyze both the uptake and evolution of hydrogen.

The hydrogen is trapped by hydrogenase in Azotobacter and recycled. The use of recycled hydrogen to generate some extra ATP may contribute to the unusually efficient ATP economy of Azotobacter.

Some rhizobia also show similar reaction besides cyanobacteria and photosynthetic bacteria which show photo-evolution of hydrogen. Non-H₂-evolving called ‘tight’ symbiosis fixes more nitrogen per unit of solar energy than ‘loose’ (H2-evolving) one. The non- leguminous associations have been “tighter” than in most of the leguminous associations.