Let us make an in-depth study of the mechanism of biological nitrogen fixation.
The biological nitrogen fixation is carried out by some bacteria, cyanobacteria and symbiotic bacteria. In symbiotic association, the bacterium provides fixed nitrogen (NH3) to the host and derives carbohydrates and other nutrients from the latter.
Biological nitrogen fixation occurs in the presence of the enzyme nitrogenase which is found inside the nitrogen fixing prokaryote. In addition to this enzyme, a source of reducing equivalents (ferredoxin (Fd) or flavodoxin in vivo), ATP and protons are required.
The overall stoichiometry of biological nitrogen fixation is represented by the following equation:
N2 + 8H+ + 8e– + 16 ATP → 2NH3 + H2 + 16 ADP + 16 Pi
The enzyme nitrogenase is in-fact an enzyme complex which consists of two metallo-proteins.
(i) Fe-protein or iron-protein component (previously called as azo ferredoxin) and
(ii) Fe Mo-protein or iron-molybdenum protein component (previously called as molybdoferredoxin). None of these two components alone can catalyse the reduction of N2 to NH3.
The Fe-protein component of nitrogenase is smaller than its other component and is an Fe-S protein which is extremely sensitive to O2 and is irreversibly inactivated by it. This Fe-S protein is a dimer of two similar peptide chains each with a molecular mass of 30-72 kDa (depending upon the micro-organism). This dimer contains four Fe atoms and four S atoms (which are labile and 12 titrable thiol groups).
The MoFe-protein component of nitrogenase is larger of the two components and consists of two different peptide chains which are associated as a mixed (α2β2 ) tetramer with a total molecular mass of 180 – 235 k Dalton (depending upon the micro-organism). This tetramer contains two Mo atoms, about 24 Fe atoms, about 24 labile S atoms and 30 titrable thiol groups probably in the form of three 24 Fe4 – S4 clusters. This component is also sensitive to O2.
i. Because nitrogenase enzyme complex is sensitive to O2, biological nitrogen fixation requires anaerobic conditions. If the nitrogen fixing organism is anaerobic than there is no such problem. But, even when the organism is aerobic, nitrogen fixation occurs only when conditions are made to maintain very low level of O2 or almost anaerobic conditions prevail inside them around the enzyme nitrogenease.
ii. Apart from N2, the enzyme nitrogenase can reduce a number of other substrates such as N2O (nitrous oxide), N3– (azide), C2H2 (acetylene), protons (2H+) and catalyse hydrolysis of ATP.
iii. Direct measurement of nitrogen fixation is done by mass spectroscopy. However, for comparative studies reduction of acetylene can be measured rather easily by gas chromatography method.
The electrons are transferred from reduced ferredoxin or flavodoxin or other effective reducing agents to Fe-protein component which gets reduced. From reduced Fe-protein, the electrons are given to MoFe-protein component which in turn gets reduced and is accompanied by hydrolysis of ATP into ADP and inorganic phosphate (Pi). Two Mg++ and 2 ATP molecules are required per electron transferred during this process.
Binding of 2 ATPs to reduced Fe-protein and subsequent hydrolysis of 2 ATPs to 2 ADP + 2 Pi is believed to cause a conformatorial change of Fe-protein which facilitates redox (reduction-oxidation) reactions. From reduced MoFe-protein, the electrons are finally transferred to molecular nitrogen (N2) and 8 protons, so that two ammonia and one hydrogen molecule are produced (see the equation and Fig. 9.4)
iv. At first glance, it might be expected that six electrons and six protons would be required for reduction of one N2 molecule to two molecules of ammonia. But, the reduction of N2 is obligatorily linked to the reduction of two protons to form one H2 molecule also. It is believed that this is necessary for the binding of nitrogen at the active site.
v. The electrons for regeneration of reduced electron donors (ferredoxin, flavodoxin etc.) are provided by the cell metabolism e.g., pyruvate oxidation.
Substantial amount of energy is lost by the micro-organisms in the formation of H2 molecule during nitrogen fixation. However, in some rhizobia, hydrogenase enzyme is found which splits H2 to electrons and protons (H2 → 2H+ + 2e–). These electrons may then be used again in reduction of nitrogen, thereby increasing the efficiency of nitrogen fixation.
Although scientists have tried to explain the mechanism of biological nitrogen fixation, but the precise pathway of electron transfer, substrate entry and product release and source of protons during biological nitrogen fixation have not yet been fully elucidated.
Formation of Root Nodules in Leguminous Plants:
The rhizobia occur as the free-living organisms in the soil before infecting their respective host plants to form root nodules. The symbiosis between rhizobia and leguminous host plant is not always obligatory. However, under conditions of limited nitrogen supply in the soil, there is elaborate exchange of signals between the two symbionts for development of symbiotic relationship.
vi. There are separate host specific genes and rhizobial specific genes which are involved in nodule formation. The host plant genes are called as nodulin or Nod genes while rhizobial genes are called as nodulation or nod genes. Some Nod factors produced by rhizobia act as signals for symbiosis.
The rhizobia migrate and accumulate in the soil near the roots of the legume plant in response to the secretion of certain chemicals such as flavonoids and be-taines by the roots. Root hairs of legume produce specific sugar binding proteins called as lectins. These lectins are activated by Nod factors to facilitate the attachment of rhizobia to the root hairs whose tips in turn become curved (Fig. 9.5 A).
Rhizobia now secrete enzymes which degrade the cell walls of root hairs at the point of their attachment for entry into the root hair. From root hairs, the rhizobia enter into the cells of inner layers of cortex through infection threads (tubular extensions of the in-folded plasma membrane produced by fusion of Golgi-derived membrane vesicles).
The rhizobia continue to multiply inside infection thread and are released into cortical cells in large numbers, where they cause cortical cells to multiply and ultimately result in the formation of nodules on the upper surface of the roots (Fig. 9.5 A & B). After their release into cortical cells, the rhizobia stop dividing and enlarge.
Electron microscopic studies have shown groups of rhizobia to the surrounded by single membranes which originate from host cell plasma membrane. The enlarged and non motile groups of bacteria inside the membranes are called as bacteroids and the membrane surrounding them as peribacterioid membrane.
The space between bacteroids and peribacteroid membrane is called as peribacteroid space. These bacteroids are aerobic and the nitrogenase enzyme is found inside them. The bacteroides lack a firm wall and are osmotically labile. In root nodule cells of Glycine max, often groups of 4 – 6 bacteroids are enclosed inside the peribacteroid membranes (Fig. 9.5 C)
The number of chromosomes in cortical cells infected by rhizobia which later develop into nodule is double the number of chromosome in other somatic cells of the legume (i.e., they are tetraploid) and seems to be pre-requisite for nodule formation. Apart from infected cells which are tetraploid, some unifected diploid cells are also found in nodule. The nodule has its own vascular system which is connected with vascular system of the root to facilitate transfer of fixed nitrogen i.e., NH3 to the host and carbohydrates and other nutrients from the host to the bacteroids.
In root nodules of leguminous plants, a red pigment- an oxygen binding heme protein which is very much similar to hemoglobin of red blood corpuscles is found. This pigment is called as leg-hemoglobin and occurs in cytosol of infected nodule cells. Leg-hemoglobin gives pinkish-red colour to the nodules. The globin part of this pigment is synthesized in host plant genome in response to the bacterial infection, while its heme portion is synthesized by bacterial genome.
Although a correlation has been found between the concentration of hemoglobin and the rate of nitrogen fixation, but this pigment does not play a direct role in nitrogen fixation. It (i) protects the nitrogenase inside the bacteroids from deterimental effect of oxygen and (ii) maintains adequate supply of oxygen to the bacteroids, so that through respiration ATPs continue to be generated which are required for nitrogen fixation.
After its formation inside bacteroids, ammonia (or NH4+) is released into cytosol of infected nodule cells where it is converted into amides (chiefly asparagine and glutamine) or ureids (chiefly allantoic acid, allantoin and citrulline). These amides or ureids are then translocated to shoots of host plant through xylem, where they are rapidly catabolized to NH4+ for entry into mainstream of ammonium assimilation.