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1. Learning Outcomes
2. Introduction
3. Fe protein
3.1 Fe-protein cycle
4. MoFe-protein
4.1 MoFe-cofactor
4.2 Role of Mo
4.2 P cluster
5. Substrate entry and product release
6. Summary
2. Introduction
The enzyme nitrogenase is found in certain bacteria and blue-green algae, which can reduce N 2 to
NH3 (nitrogen fixation). Some of these bacteria are free-living while others are symbiotic (in the
anaerobic environment of roots of legume plants). This bacterial reaction is the key step in the
nitrogen cycle, which maintains a balance between two reservoirs of the nitrogen compounds.
Nitrogen-fixing bacteria catalyze dinitrogen reduction in forming two ammonia molecules,
resulting the major contribution of fixed nitrogen to the biogeochemical nitrogen cycle. Although,
the reduction of dinitrogen still remains one of the today’s biggest challenges in chemistry. As a
result, substantial efforts have been applied to understanding the mechanism of bacterial
nitrogenases. The transient interaction of two component proteins are involved in the reduction of
N2 by this enzyme, designated the Fe protein and the MoFe protein, and minimally requires
sixteen MgATP, eight protons and electrons.
Four types of nitrogenases are known till date, each having a different combination of metals at
the active site. But, the most abundant nitrogenase type is the Mo-nitrogenase, which has an
active site called FeMo-cofactor. This cofactor contains Mo along with Fe, S, (R)-homocitrate,
and a light atom of unknown identity called X.
The ideal reaction catalyzed by the Mo-dependent nitrogenase can be represented by the chemical
equation (eqn 1):
The Mo-nitrogenase is
composed of two-component
The Mo-nitrogenase is composedmetalloproteins
of two-component metalloproteins commonly called as the iron
commonly
protein and the molybdenum-ironcalled
protein. Their structures either individually or in the complex
as the iron protein and
form are determined by X-ray the
crystallography that hasprotein.
molybdenum-iron provided detailed models of the three
metal clusters present in nitrogenase. The nitrogen fixation by nitrogenase contains a complex
interaction between the two protein components, MgATP electrons and protons. Understanding
the molecular details of this multi-step and multi-component system is difficult.
Figure 1: Structure of M-cluster of Mo-nitrogenase
Physiologically, reduction of Fe protein depends upon the organism, with reduced ferredoxin or
flavodoxin being the most common electron donor. Lots of questions still remain unanswered
regarding the mechanistic details of the Fe protein cycle. For instance, the nature of the
interaction between the nucleotide binding sites in the Fe protein and MoFe protein interface
remain obscure. The reason behind undetectable display rates for MgATP hydrolysis in Fe
protein prior to association with the MoFe protein. How the hydrolysis of MgATP gets activated,
once it bound to the MoFe protein. Likewise, very little is known about how MgATP binding and
hydrolysis within the Fe protein is communicated into the MoFe protein. This type of
communication is suggested by the observation that the Fe protein is the only known reductant
for the MoFe protein that will support substrate reduction. The capacity of small-molecule
4. MoFe-protein
The MoFe protein is a heterotetramer (Mr≈ 250,000) conatining two pairs of metalloclusters,
named P-cluster ([8Fe-7S]) and FeMo-cofactor ([7Fe-Mo-9S-homocitrate-X]) (Figure 2).
4.1 FeMo-cofactor
A [4Fe-3S] subcluster is connected to a [3Fe-Mo-3S] subcluster by an atom X at one corner and three bridging inorganic sul
FeMo-cofactor is anchored to the MoFe protein by α-275Cys to an iron atom at one end and α-
442His to the Mo atom at the other end (Figure 2).
Certain mutant strains that are deficient in FeMo-cofactor biosynthesis are inactive for reduction of all substrates, but gets activ
MoFe protein that has a structurally altered FeMo-cofactor, wherein citrate substitutes for homocitrate, display altered catalytic
Amino acid substitutions in the FeMo-cofactor binding environment bring out changes in the catalytic and spectroscopic prope
In CO-inhibited MoFe protein, ENDOR spectroscopy demonstrates that the CO binds to FeMo-cofactor, not to the P cluster.
4.3 P-cluster
These are [8Fe-7S] clusters, they are responsible for electron transfer between the Fe protein and
the substrate reduction site FeMo-cofactor due to the following:
The P-cluster are made from two linked [4Fe-4S] subclusters having common bridging µ6-sulfide
at one corner(figure.3). Cysteinate-S ligands is the only known naturally occurring [Fe-S] cluster
containing serinate-O (β-188Ser) and amideN (α-88Cys) ligands. X-ray crystallographic studies
of two different oxidation states of the MoFe protein tells that P-clusters undergo structural
rearrangement upon oxidation (pOX) from the resting state (dithionite reduced; pN), one
consequence of which is the displacement of both the serinate-O and amideN ligands from pN.
There is very less information about the oxidation states that the P-cluster accesses during
turnover. According to this information a practical working model of P-cluster function is made.
According to which, firstly one or two electron is transferred from the P-cluster to FeMo-cofactor
upon association of the Fe protein with the MoFe protein, that is followed by re-reduction of the
oxidized P-cluster by electron transfer from the Fe protein.
During catalysis, the FeMo-cofactor should accept eight electrons, whereas only one
electron is delivered during each event of Fe protein binding.
The subscript denotes the number of electrons (and protons) added to the MoFe protein (E)
(Figure 4, bottom cycle).
Thus, MoFe protein proceeds through states from E 0 to E8 during N2 fixation before it returns
back to the resting state (E0).
The 1-electron Fe protein cycle and 8-electron MoFe-protein cycle can be conceived of as
interlocking, with the Fe protein cycle (Figure 4, top cycle) driving the MoFe protein (Figure 4,
bottom cycle) to successively reduced states.
This model for the nitrogenase mechanism depicts several important observations regarding the
mechanics of catalysis.
For instance, it is known that three or four electrons must accumulate within the MoFe protein
before N2 binds (E3 or E4 states).
Moreover, a stoichiometric quantity of H 2 is evolved, when N2 binds to the MoFe protein.
Besides, reducing N2 and protons, nitrogenase has the ability to reduce a wide variety of small
unsaturated molecules, such as azides, nitrous oxide, nitriles, isonitriles, and alkynes.
The reduction of the acetylene (C 2H2) to ethylene (C2H4) is the most commonly used method for
monitoring nitrogenase activity.
Even though both acetylene and N2 have potential to bind to the same site on FeMo-cofactor, it is
worth mentioning that acetylene binds to a less-reduced E state (E 2) than does N2 (E3, E4).
Therefore, when these two compounds are present, acetylene appears to be a non-competitive
inhibitor of N2 reduction.
Although the FeMo-cofactor is buried quite below the surface of protein and no permanent
channels are there between the MoFe-cofactor and the protein, but there are two clefts which can
be used for the substrate entering and release of product and/or transfer of H30+ to the active site.
The first cleft is made up of five stretches of polypeptide chain (a255-a275, a279- a301, a338-a359, a363-a378, and a422-a43
Both of these clefts have funnel like shape and are near to one of the putative Fe-protein subunit
binding sites. However, these two clefts are not wide enough to allow free diffusion of either
substrate or product or H3O+. Therefore, it seems reasonable to assume that some conformational
changes must be there which provides diffusion of ligands into and away from the FeMo-
cofactor, similar to the case of binding and release of oxygen to the buried hemes in globins.
The details of the interaction between various substrates and the FeMo-cofactor could provide
understanding in the catalytic function of nitrogenase. Therefore, it becomes necessary to
understand different modes of interactions between them. There are many possibilities by which
substrate can bind to one or more of the Fe, Mo, and/or S site. Of all, the six trigonally
coordinated iron sites of the FeMo-cofactor are particularly fascinating for the substrate binding.
Based on the FeMo-cofactor structure, three different types of binding modes of substrates to
these coordinatively unsaturated irons may be envisioned:
End-on fashion: Substrates could bind in an end-on fashion to one of the six central iron
sites which are bridged by non protein ligands. It is also conceivable that some substrates
could displace the bridging ligand Y, if this ligand is not a sulfur, and interact
simultaneously with the two adjacent iron sites.
The central cavity: Some small substrates, such as N 2 and/or H+, could occupy the
central cavity in the FeMo-cofactor, thereby replacing the weak iron-iron bonds with Fe-
substrate bonds.
The exterior surface: Substrates could bind to the exterior surface of the FeMo-cofactor.
Three sets of cyclic eight-membered rings occur on the exterior surface of the FeMo-
cofactor, consisting of alternating S(Y) and Fe sites.
The existence of multiple potential substrate-binding sites in the FeMo-cofactor could be linked
with the noncompetitive kinetics observed between N 2 and other substrates.
The complete structural model for nitrogen fixation is shown in figure 5.
6. Summary