Mechanism of Biological Nitrogen Fixation: Navigation

5/30/2019 Mechanism of Biological Nitrogen Fixation
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Mechanism of
Biological Nitrogen
Fixation
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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.
Leaf: Definition, Parts and Types (With Diagram)|
Botany
Biological nitrogen fixation occurs in the
presence of the enzyme nitrogenase which is Read Next Story
found inside the nitrogen fixing prokaryote.

www.biologydiscussion.com/plant-physiology-2/nitrogen-metabolism/mechanism-of-biological-nitrogen-fixation/22810 1/23
In addition to this enzyme, a source of

reducing equivalents (ferredoxin (Fd) or
flavodoxin in vivo), ATP and protons are
required.
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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
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Botany
Read Next Story
(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 Leaf:
peptide chains
Definition, Parts and Types (With Diagram)|
Botany
which are associated as a mixed (α2β2 )
tetramer with a total molecular mass of 180 Read Next Story
– 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 transferredLeaf:

from reducedParts and Types (With Diagram)|
Definition,
Botany
ferredoxin or flavodoxin or other effective
reducing agents to Fe-protein component Read Next Story
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 obligatorilyBotany
linked to the
reduction of two protons to form one H2
Read Next Story
molecule also. It is believed that this is
necessary for the binding of nitrogen at the

active site.
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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 triedLeaf:

to explain the
Definition, Parts and Types (With Diagram)|
mechanism of biological nitrogen
Botanyfixation,
but the precise pathway of electron transfer,

Read Next Story
substrate entry and product release and
source of protons during biological nitrogen

fixation have not yet been fully elucidated.
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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.Botany
The host plant
genes are called as nodulin or Nod genes
Read Next Story
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).

Botany
Rhizobia now secrete enzymes which Read Next Story
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 exten-
sions 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 ulti-
mately 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.

Botany
Read Next Story
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.
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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)
Botany
Read Next Story
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
Leaf: in Parts and Types (With Diagram)|
Definition,
Botany
cytosol of infected nodule cells. Leg-
hemoglobin gives pinkish-red colour to the Read Next Story
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) main-
tains 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.
Related Articles:
1. Fixation of Atmospheric Nitrogen
Botany |
Enzyme System
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5/30/2019 Mechanism of Biological Nitrogen Fixation

Let us make an in-depth study of the

The biological nitrogen fixation is carried

found inside the nitrogen fixing prokaryote.

In addition to this enzyme, a source of

The overall stoichiometry of biological

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

The enzyme nitrogenase is in-fact an enzyme

(i) Fe-protein or iron-protein component

ADVERTISEMENTS: Leaf: Definition, Parts and Types (With Diagram)|

Read Next Story

(ii) Fe Mo-protein or iron-molybdenum

The Fe-protein component of nitrogenase is

The MoFe-protein component of

– 235 k Dalton (depending upon the micro-

organism). This tetramer contains two Mo

i. Because nitrogenase enzyme complex is

ii. Apart from N2, the enzyme nitrogenase

iii. Direct measurement of nitrogen fixation

The electrons are transferredLeaf:

which gets reduced. From reduced Fe-

protein, the electrons are given to MoFe-

Binding of 2 ATPs to reduced Fe-protein and

iv. At first glance, it might be expected that

necessary for the binding of nitrogen at the

v. The electrons for regeneration of reduced

Substantial amount of energy is lost by the

Although scientists have triedLeaf:

but the precise pathway of electron transfer,

source of protons during biological nitrogen

Formation of Root Nodules in

The rhizobia occur as the free-living

vi. There are separate host specific genes

nodulation or nod genes. Some Nod factors

The rhizobia migrate and accumulate in the

Leaf: Definition, Parts and Types (With Diagram)|

Rhizobia now secrete enzymes which Read Next Story

degrade the cell walls of root hairs at the

point of their attachment for entry into the

The rhizobia continue to multiply inside

Leaf: Definition, Parts and Types (With Diagram)|

Read Next Story

Electron microscopic studies have shown

The space between bacteroids and

Read Next Story

The number of chromosomes in cortical

In root nodules of leguminous plants, a red

synthesized in host plant genome in

Although a correlation has been found

After its formation inside bacteroids,

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