Special Features of Diazotrophs
Sites of N2
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
. Structure of nitrogenase is given in Fig. 11.1. It consists of two brown metalloproteins whose joint action is essential for reduction of N2
. 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.
Fig. 11.1. Diagrammatic structure of nitrogenase. A, component I; B, component II; C, nitrogenase complex
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).
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 :
+12 Mg ATP + 6(H+
+ 12 (Mg ADP + Pi) (Johansson et al,
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).
Fig. 11.2. Reuse of H2 by hydrogenase.
On the basis of inhibitor studies, Hwang et al
(1973) have proposed 5 sites for nitrogenase complex such as (i)
acetylene site, (iii)
azide, cyanide and methyl isocyanide sites, (iv)
Co site, and (v)
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,
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).
Fig. 11.3. Nitrogen fixation by nitrogenase complex (A) and mechanism of N2 conversion by component I (B).
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+
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 :
|H2 + S
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
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
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.
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