Nitrogen fixation mechanism and genes involved in nitrogen fixation

1. Nitrogen fixation mechanism and
genes involved in Nitrogen fixation
Dr. Aswartha Harinath Reddy M.Sc, Ph.D.
Department of Life Sciences
Srikrishnadevaraya University
Anantapur –A.P. India

2.  Biological Nitrogen fixation is a process by which nitrogen in
the Earth's atmosphere is converted into ammonia (NH3) or
other molecules available to living organisms.
 Nitrogen fixation is carried out naturally in the soil by
nitrogen fixing bacteria such as Azotobacter.
 Some nitrogen-fixing bacteria have symbiotic relationships
with some plant groups, especially leguminous plants
(Rhizobia).

3.  Biological nitrogen fixation was discovered by the Dutch
microbiologist Martinus Beijerinck.
 Biological nitrogen fixation (BNF) occurs when atmospheric
nitrogen is converted to ammonia by an enzyme called a
nitrogenase.
 All biological nitrogen fixation is done by way of metalloenzyme
called nitrogenase.
 These enzymes contain iron, often with a second metal, usually
molybdenum as co-factors.

4.  Biological nitrogen fixation can be represented by the
following equation, in which two moles of ammonia are
produced from one mole of nitrogen gas, at the expense of
16 ATP molecules and a supply of eight electrons and
protons (hydrogen ions):
N2 + 8H+ + 8e- + 16 ATP = 2NH3 + H2 + 16ADP + 16 Pi
 The process is coupled to the hydrolysis of 16 ATPs and is
accompanied by the co-formation of one molecule of H2 and
two molecules of Ammonia.

5.  Enzymes responsible for nitrogen fixation are very susceptible to
atmospheric oxygen.
 For this reason, many bacteria cease production of the enzyme in
the presence of oxygen.
 Di-azotrophs are a diverse group of prokaryotes that includes
Cyanobacteria, green sulfur bacteria, along with Azotobacteraceae,
rhizobia and Frankia.
 Diazotrophs are bacteria and archaea that fix atmospheric nitrogen
gas into a more usable form such as ammonia.

6.  The enzyme nitrogenase is an enzyme complex which consists
of two metallo-proteins or two complexes.
 (i) Complex-I (Fe-protein complex).
 (ii) Complex-II (Fe Mo-protein complex).

7. Fe-protein complex (Dinitrogenase Reductase):
 The Fe-protein component of nitrogenase is smaller than its other
component and is an Fe-S protein which is extremely sensitive to
O2.
 Dinitrogenase reductase is an α2 dimer of the nifH gene product,
contains four Fe atoms and four S atoms.
 Dinitrogenase reductase exists in the oxidized ([Fe4S4] 2+) and
two electron reduced ([Fe4S4]0) forms.
 Dinitrogenase reductase binds two molecules of Mg and two
molecules of ATP.

8. Iron-molybdenum protein complex (Dinitrogenase).
 Dinitrogenase is an α2β2 tetramer of the nifD and nifK
gene products and has a molecular weight of
approximately 240 kDa.
 This tetramer contains two Mo atoms, about 24 Fe atoms,
about 24 S atoms.
 This component is also sensitive to O2.

9.  Pyruvate functions both as an electron donor and an energy source.
 Since all nitrogen-fixing microorganisms contain hydrogenase, this
enzyme system in cells catalyzes the transfer of electrons from
pyruvate to Ferredoxin.
 The electrons are transferred from reduced Ferredoxin to Fe-S
protein component which gets reduced, and is accompanied by
hydrolysis of ATP.
 Two Mg++and 2 ATP molecules are required per electron
transferred during this process.
Mechanism:

10.  From reduced Fe-protein component (Complex-I ), the elec-
trons are given to MoFe-protein component (Complex-II)
which in turn gets reduced.
 From reduced MoFe-protein component, the electrons are
finally transferred to molecular nitrogen (N2), so that two
ammonia and one hydrogen molecule are produced.

11. 16ATP 16 ADP+Pi
N2 + 8H+ + 8e- + 16 ATP = 2NH3 + H2 + 16ADP + 16 Pi
Complex 1 Complex 2

12. NH3
NH3
NH3

13. Genes involved in N2 Fixation:
 N2-fixation requires the coordinate interaction of nif genes
present in rhizobia.
 For example in the free-living Azotobacter, at least 20 nif
genes are organized in about 8 operons.
 Nitrogen fixation in symbionts and free-living microbes is
catalyzed by nitrogenase, an enzyme complex encoded by
nifD&K (Complex II) and nifH genes (Complex I).
 The FeMo cofactor (FeMo-Coo) is required for activation of
the complex I and II. This is assembled from nifB, N, V and
nifE genes.

15.  In most systems the regulation of all nif genes is controlled by NifA
(a positive regulator of transcription) and NifL (the negative
regulator).
 Environmentally, nif gene expression is regulated by both oxygen
and nitrogen levels.
 For example, elevated soil ammonia (NH3 or NH4) concentrations
allows NifL to act as a negative controller and prevent the
experession of nif genes.
 In addition, elevated O2 concentrations also prevent the expression
of nif genes.

17. nifH: Dinitrogenase reductase, Also is required for FeMo-Co biosynthesis.
nifD: α subunit of dinitrogenase. Forms an α2 β2 tetramer.
nifK: β subunits of dinitrogenase.
nifT : Unknown
nifE: Forms an α2 β2 tetramer. Required for FeMo-co synthesis.
nifN: Required for FeMo-co synthesis.
nifx: Involved in FeMo-co synthesis.
nifU: Involved in mobilization of Fe-S cluster synthesis and repair.
nifL: Negative regulatory element.
nifA: Positive regulatory element.
nifB: Required FeMo-co synthesis.
nifJ : Pyruvate flavodoxin (ferredoxin)
12genes
Nif genes products and their role in Nitrogen fixation:

18. 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.
 Rhizobial specific genes which are involved in nodule formation
called nodulation or nod genes. Some Nod factors produced by
rhizobia act as signals for symbiosis.
 Lately, there are more than 65 nodulation genes have been
identified in rhizobia.

19.  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
organic acids by the roots.
 Root hairs of legume produce specific sugar binding
proteins called as lectins.
 These lectins facilitate the attachment of rhizobia to
the root hairs whose tips in turn become curved.

20.  From root hairs, the rhizobia enter into the cells of inner
layers of cortex through infection threads.
 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.
 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.

22.  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.

23.  Leg-hemoglobin protects the nitrogenase inside the
bacteroids from deterimental effect of oxygen.
 It maintains adequate supply of oxygen to the
bacteroids, so that through respiration ATPs continue
to be generated which are required for nitrogen
fixation.

24. Thank You………