The document discusses nitrogenase enzymes, which are produced by certain bacteria and are responsible for reducing nitrogen gas to ammonia. It notes that nitrogen fixation is essential for life. The summary describes that nitrogenase consists of two components - the MoFe protein that catalyzes nitrogen reduction and the Fe protein that supplies electrons. The mechanism involves electrons flowing from ATP to the Fe-S cluster and then to the FeMo cofactor where nitrogen is reduced to ammonia. Key bacteria that synthesize nitrogenase include rhizobia, cyanobacteria, and azotobacter.

1. SRI PARAMAKALYANI COLLEGE
Reaccredited with B Grade with a CGPA of 2.71 in the second cycle of NAAC
affiliated to manonmanium sundaranar university, Tirunelveli.
ALWARKURICHI – 627 412
Post graduate and Research Centre – Department of Microbiology
(Government aided)
ACADEMIC YEAR 2021 – 2022
I SEM CORE: PHYSIOLOGY AND METABOLISM
UNIT IV
NITROGENASE ENZYME
SUBMITTED BY,
K. RAMKUMAR
REG NO: 20211232516122
I M.SC MICROBIOLOGY
SUBMITTED TO
GUIDE: DR.S.VISWANATHAN
ASSISTANT PROFESSOR AND
HEAD OF DEPARTMENT

2. Synopsis
 Introduction
 Classification
 Structure
 Nitrogenase
 Mechanism
 Organisms synthesize

3. Introduction
 Nitrogenase are enzymes, that are produced by certain bacteria (such as cyanobacteria and
rhizobacteria).
 These enzymes are responsible for the reduction of nitrogen (N2) to ammonia (NH3).
 Nitrogenases are the only family of enzymes known to catalyze this reaction, which is a key
step in the process of Nitrogen fixation.
 Nitrogen fixation is required for all forms of life, with nitrogen being essential for
the biosynthesis of molecules (nucleotides, amino acids) that create plants, animals and other
organisms.
 They are encoded by the Nif genes or homologs. They are related to protochlorophyllide
reductase.

4. Classification
Molybdenum nitrogenase (Mo)
Vanadium nitrogenase (V)
Iron-only nitrogenase.

5. Structure
 Although the equilibrium of ammonia from molecular hydrogen and
nitrogen has an overall negative enthalpy of reaction, the activation
energy is very high.
 Nitrogenase acts as a catalyst.
 Consists of two components:
I. The heterotetrameric MoFe protein, a nitrogenase which uses the
electrons provided to reduce
II. The homodimeric Fe-only protein, the reductase which has a
high reducing power and is responsible for the the supply of electrons.

6. Structure:

7. Nitrogenase
 The MoFe protein is a heterotetramer consisting of two α subunits and two
β subunits, with a mass of approximately 240-250kDa.
 The MoFe protein also contains two iron–sulfur clusters, known as P-
clusters, located at the interface between the α and β subunits and two FeMo
cofactors, within the α subunits.
 The oxidation state of Mo in these nitrogenases was formerly thought
Mo(V), but more recent evidence is for Mo(III).
 Molybdenum in other enzymes is generally bound to molybdopterin as fully
oxidized Mo(VI).
The core (Fe8S7) of the P-cluster takes the form of two [Fe4S3] cubes linked
by a central carbon atom. Each P-cluster is covalently linked to the MoFe
protein by six cysteine residues.

8.  Each FeMo cofactor (Fe7MoS9C) consists of two non-identical clusters: [Fe4S3] and
[MoFe3S3], which are linked by three sulfide ions. Each FeMo cofactor is covalently linked
to the α subunit of the protein by one cysteine residue and one histidine residue.
 Electrons from the Fe protein enter the MoFe protein at the P-clusters, which then
transfer the electrons to the FeMo cofactors. Each FeMo cofactor then acts as a site for
nitrogen fixation, with N2 binding in the central cavity of the cofactor.

9. Mechanism
 Nitrogenase is an enzyme responsible for catalyzing nitrogen fixation,
which is the reduction of nitrogen (N2) to ammonia (NH3) and a process vital
to sustaining life on Earth.
 There are three types of nitrogenase found in various nitrogen-fixing
bacteria: molybdenum (Mo) nitrogenase, vanadium (V) nitrogenase, and
iron-only (Fe) nitrogenase.
 Molybdenum nitrogenase, which can be found in diazotrophs such
as legume-associated rhizobia, is the nitrogenase that has been studied the
most extensively and thus is the most well characterized.
 The crystal structure and key catalytic components of molybdenum
nitrogenase extracted from Azotobacter vinelandii.
 Vanadium nitrogenase and iron-only nitrogenase can both be found in select
species of Azotobacter as an alternative nitrogenase.

10. Function:

11.  Equations 1 and 2 show the balanced reactions of nitrogen fixation in
molybdenum nitrogenase and vanadium nitrogenase respectively.
N2 + 14 H+ + 12 e− + 40 MgATP → 2 NH4
+ + 3 H2 + 40 MgADP + 40 Pi
N2 + 8 H+
+ 8 e−
+ 16 MgATP → 2 NH3 + H2 + 16 MgADP + 16 Pi

12.  All nitrogenases are two-component systems made up of Component I (also
known as dinitrogenase) and Component II (also known as dinitrogenase
reductase).
 Component I is a MoFe protein in molybdenum nitrogenase, a VFe protein
in vanadium nitrogenase, and a Fe protein in iron-only nitrogenase.
 Component II is a Fe protein that contains the Fe-S cluster, which transfers
electrons to Component I.
 Component I contains 2 key metal clusters: the P-cluster, and the FeMo-
cofactor (FeMo-co) . Mo is replaced by V or Fe in vanadium nitrogenase and
iron-only nitrogenase respectively.
 During catalysis, electrons flow from a pair of ATP molecules within
Component II to the Fe-S cluster, to the P-cluster, and finally to the FeMo-
co, where reduction of N2 to NH3 takes place.

13. Picture:

14. Organisms synthesize
 Symbiotic bacteria – Example: Rhizobium, spirillum, Frankia
 Non symbiotic bacteria – Example: cyanobacteria, azotobacter, Green s
bacteria.