Special Features of Diazotrophs

Sites of N₂ Fixation
Many cyanobacteria capable of fixing nitrogen are filamentous and contain pale and thick walled cell (intercalary, lateral or terminal, single or in chains) called heterocyst. These are the sites of nitrogen fixation. Heterocysts are formed in the absence of utilizable combined nitrogen, such as ammonia, because it inhibits heteroyst differentiation and N₂ fixing enzyme, the nitrogenase. As soon as cyanobacteria growing on ammonia-supplemented media are transferred to nitrogen medium, both heterocysts and nitrogenase develop parallelly. Besides, some strains, high light intensity inhibits N₂ fixation. Heterocyst lacks oxygen evolving photosystem II, ribulose biphosphate carboxylase and may lack or have reduced amount of photosynthetic biliproteins (Wolk, 1982). Chlorophyll-a is present in the heterocysts. Wall of heterocyst contains O₂ binding glycolipids which, together with respiratory consumption, maintain the anaerobic conditions (highly reduced atmosphere) necessary for N₂ fixation (Bothe, 1982). In contrast, in vegetative cells adjacent to heterocysts, both photosystem I and II are present, therefore, oxygen evolution takes place by these cells.

In other group of cyanobacteria such as Lyngbya, Oscillatoria, Plectonoma, and bacteria where heterocyst does not develop, N₂-fixation process takes place in internally organized cells having reduced conditions.

Root nodules developed by Rhizobium (in leguminous plants) and Frankia (in non-leguminous plants) are the site of nitrogen fixation (see Symbiotic N₂ fixation). However, lichens (a symbiotic structure formed by cyanobacteria and fungi) are the site of N₂ fixation in lower group of microorganisms.

Presence of Nitrogenase and Reductants
All diazotrophs possess an enzyme nitrogenase which helps in conversion of N₂ to NH₃. Structure of nitrogenase is given in Fig. 11.1. It consists of two brown metalloproteins whose joint action is essential for reduction of N₂ to IsfH₃. Component I i.e. molybdo-ferro-protein (Mo-Fe-protein) which is also known as nitrogenase has a molecular weight of about 2.2 x 10⁵ dalton. Nitrogenase contains molybdenum (2 atoms/mol), iron (32 atoms/mol) and a sulphur (30 atoms/mol). It is a larger unit than component II and non-sensitive to cold but losses activity at 0°C. On the other hand component II, nitrogenase reductase; is a smaller unit than the former one.

It contains ferro-protein (Fe-protein) and has a molecular weight of about 5 x 10⁴ dalton. It contains iron (4 atoms/ mol) and sulphur (4 atoms/mol), and is less stable than the component I (Sasson, 1984).

Thus, nitrogenase is an equilibrium mixture of Mo-fe-protein and Fe-protein in the ratio 1: 2 (Fig. 11.1).
Mo-Fe-Protein+2 (Fe-protein)  Nitrogenase

Whenever energy is needed, ATP undergoes enzymatic hydrolysis to form ADP + Pi. Mg⁺⁺ functions as catalyst. Overall reaction clayed by nitrogenase may be written as below :
N₂+12 Mg ATP + 6(H⁺ + e^-)2NH₃ + 12 (Mg ADP + Pi) (Johansson et al, 1983).

In Rhodospirillum rubrum the physiological donors (e.g. ferredoxin I and II) of nitrogenase has been demonstrated to transfer reducing equivalent from the illuminated chloroplast to crude preparation of R. rubrum nitrogenase (Yoch and Arnon, 1975). Recently, it has been shown that pyruvate- 2-oxoglutartate and to a lesser extent, oxaloacetate supported nitrogenase activity in crude extract of R. rubrum (Ludden and Burris, 1981).

In addition to reduction of nitrogenase to ammonia, nitrogenase catalyses a number of other substrates. It has been found that nitrogenase isolated from R. rubrum catalyzed the reduction of acetylene, protons, cyanide, isocyanide and azide; all of which behaved as the substrates of nitrogenase from non-phototrophic bacteria (Munson and Burris, 1969).

On the basis of inhibitor studies, Hwang et al (1973) have proposed 5 sites for nitrogenase complex such as (i) N₂ and N₂ sites, (ii) acetylene site, (iii) azide, cyanide and methyl isocyanide sites, (iv) Co site, and (v) H⁺ site. However, the allocation of electrons to these substrates depends on their relative concentrations and the electron efflux through the complex. At low electron efflux, N₂ competes poorly with acetylene and H⁺ as a substrate but at high electron efflux N₂ becomes a very effective substrate as the dominant electron acceptor. The electron efflux can be altered by changing the concentration of Mg ATP or the ratio of the Fe-protein to Mo-Fe-protein (Hageman et al, 1980).

Presence of Hydrogenase
Many diazotrophs evolve hydrogen (H₂) during N₂ fixation which in turn inhibits N₂ fixation reaction. To get protection from inhibition by H₂ many diazotrophs possess, an enzyme hydrogenase to recycle H₂ produced by nitrogenase (Fig. 11.2). Reutilization of H₂ produces more ATP and improves the efficiency of N₂ fixation (Johansson et al, 1983). For the first time, utilization of H₂ by purple bacteria was discovered by Roelofsen (1935).  
Present knowledge indicates that the hydrogenase of purple bacteria is membrane-bound or at least membrane-associated.

It is cold labile and very sensitive to O₂. The purified enzyme has a molecular weight of about 6,500 and most probably consists of a single polypeptide chain with 4 iron atoms and 4 acid labile sulphur atoms per molecule. It does not use NAD⁺ or NADP⁺ as electron acceptors.

Hydrogenase combines with H₂ and reduces into a number of substrates (S) to form products (SH₂) or liberates H₂ from the reduced compounds as below :

	Hydrogenase
H2 + S		SH2

Hydrogen production and N₂ fixation have shown the close relationship in some of studies, where both the processes are catalyzed by the same enzyme or enzyme complex (e.g. Azotobactor vinelandii) (Bulen et al., 1965). H₂ evolved by nitrogenase of A. chroococcum is diffused very quickly to be able to inhibit N₂ reduction. Therefore, H₂ production is an intrinsic activity of nitrogenase. Possession of this activity has been suggested to function as a safety valve through which the cell can dispose excess reducing power (Gest, 1972).

When substrate differs from N₂, nitrogenase of non-phototrophic bacteria catalyses the reduction of these viz., acetylene, protons, cyanide, isocyanide and azide, and produces H₂ which is of physiological importance. Some electrons are diverted to produce H₂ in vitro even in the presence of nitrogen. This phenomenon is attributed to Mo-Fe-protein being less reduced when present in excess amount or low concentration of ATP (Hageman and Burris, 1980).

Ammonium salts are required for vegetative growth but in excess amount, it inhibits nitrogenase and declines N₂ fixation process. It is obvious that ammonia is generated in microbial cells from N₂ and at the same time is inhibitory to nitrogenase activity. Therefore, to continue N₂ fixation process, the necessary step is to convert ammonia rapidly to organic nitrogen compounds or be removed from intracellular site of N₂ metabolism (Alexander, 1977).

Presence of Self Regulatory Systems
In diazotrophs nitrogenase activity is regulated by a complex mechanism developed in different microorganisms in different ways. Oxygen is required by aerobic bacteria and evolved by cyanobacteria. However, O₂ inhibits nitrogenase activity. To carry out N₂ fixation process efficiently, diazotrophs possess certain physiological adaptations as the defense mechanisms so that they can protect nitrogenase and carry out the N₂ fixation process.

Azotobacters are among the most O₂ tolerant free living diazotrophs. The molecular features .that allow this tolerance to O₂, include an O₂ excluding and protective protein that complexes with nitrogenase in vitro during the period of O₂ stress (Haaker and Keeger, 1977; Robson, 1979). In Azotobacters protection is given to cell by their high respiratory rate. Due to which O₂ is prevented from reaching the site of nitrogenase reaction.

Another important feature is the presence of an alternative system (in A. vinelandii) for reducing N₂ to NH₃ when organisms are grown under conditions of Mo-deprivation. This system does not require the conventional nif HDKgenes which encode nitrogenase Fe-protein and Mo-Fe-protein.

In cyanobacteria, creation of O₂ tension is remarkable as the photoautotrophs evolve O₂ which inhibits nitrogenase activity. Hydrolysis of ATP in N₂ fixing system provides an anhydrous atmosphere around the active site of nitrogenase and keeps enzyme in its most active form and activates electrons.

In heterocystous cyanobacteria, the compartmentation of photosynthesis and N₂ fixation takes place. N₂ is reduced only in heterocysts where photosynthetic systems (thylakoids) are in reduced amount or absent, therefore, nitrogenase is not inhibited by O₂ evolved during photosynthesis.

The heterocysts lack O₂ evolving photosystem II apparatus, ribulose biphosphate carboxylase and may lack or have reduced amount of photosynthetic biliproteins (Wolk, 1982). The walls of heterocyst contain O₂ binding glycolipids which, together with respiratory oxygen consumption, maintain the conditions necessary for N₂ fixation (Bothe, 1982). In contrast, the vegetative cells of filaments/trichomes contain both photosystem I and II and, therefore, evolve oxygen.

N₂ fixation has been reported in non-heterocystous species e.g. Oscillatoria and Lyngbya where such cells are located in the centre of the colony. These cells do not produce O₂ photosynthetically and thickened specialized cell walls as in heterocysts. However, N₂ fixation takes place in the same way as in heterocysts. Presence of leghaemoglobin in root nodules denotes regulation of O₂ tension and carry out N₂ fixation process (see Mechanism of N₂ fixation in root nodules).