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1. Presented By:
HITESH CH DAS
M.Sc. CHEMISTRY
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2.  Introduction
 Structural classification
 [NiFe] hydrogenase
 [FeFe] hydrogenases
 [Fe]-only hydrogenase
 Mechanism
 Functional models
 Applications
 Biological hydrogen production
 Hydrogenase-based biofuel cells
 CONCLUSION
 References
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3. Hydrogen uptake is coupled to reduction of electron acceptors
such as oxygen, nitrate, sulfate, carbon dioxide (CO2), and
fumarate.
Ferredoxin [FNRs], cytochrome c3, and cytochrome c6 can act as
physiological electron donors or acceptors for hydrogenases.
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4. Based on the structure and the metal they contain in active site
Three types of hydrogenases:
[FeFe] hydrogenase - contains two Fe in active site
[NiFe] hydrogenase – contains Ni and Fe
[Fe]-only hydrogenase – contains one Fe
 [NiFe] and [FeFe] hydrogenases have an active site with metal
and a few Fe-S clusters that are buried in protein
 While [Fe]-only hydrogenase has a mononuclear Fe active site
and no Fe-S clusters
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5. Friday, October 18, 2019 5
Ni-Fe hydrogenase Fe-Fe hydrogenase Fe only hydrogenase

6.  The [NiFe] hydrogenases are heterodimeric proteins
consisting of small and large subunits. The small subunit
contains three iron-sulfur clusters.
 while the large subunit contains the active site, a nickel-iron
centre which is connected to the solvent by a molecular
tunnel.
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Source: dx.doi.org/10.1021/cr4005814 | Chem. Rev. 2014, 114, 4081−4148

7.  The hydrogenases containing a di-iron center with a
bridging dithiolate cofactor are called [FeFe] hydrogenases.
 In contrast to [NiFe] hydrogenases, [FeFe] hydrogenases are
generally more active in production of molecular hydrogen.
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Source: dx.doi.org/10.1021/cr4005814 | Chem. Rev. 2014, 114, 4081−4148

8.  Unlike the other two types, [Fe]-only hydrogenases are
found only in some hydrogenotrophic methanogenic
archaea.
 They also feature a fundamentally different enzymatic
mechanism in terms of redox partners and how
electrons are delivered to the active site.
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10.  The hydrogenases containing a di-iron center with a bridging dithiolate cofactor are
called [FeFe] hydrogenases
 There are three class of [FeFe] hydrogenases
 cytoplasmic, soluble, monomeric hydrogenases, found in strict anaerobes such as
Clostridium pasteurianum and Megasphaera elsdenii. They catalyse both H2 evolution
and uptake.
 soluble, monomeric hydrogenases, found in chloroplasts of green alga Scenedesmus
obliquus, catalyses H2 evolution. The [Fe2S2] ferredoxin functions as natural electron
donor linking the enzyme to the photosynthetic electron transport chain
 In contrast to [NiFe] hydrogenases, [FeFe] hydrogenases are generally more active in
production of molecular hydrogen.
 Turnover frequency (TOF) in the order of 10,000 s−1 have been reported in literature for
[FeFe] hydrogenases from Clostridium pasteurianum.
 This has led to intense research focusing on use of [FeFe] hydrogenase for sustainable
production of H2.
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12.  found in methanogenic Archaea
 contains neither nickel nor iron-sulfur clusters but an iron-
containing cofactor .
 recently characterized by X-ray diffraction.
 Different from the two hydrogenase as its function is the
reversible reduction of methenyl-H4MPT+ to methylene-
H4MPT.
 also known as H2-forming
methylenetetrahydromethanopterin (methylene-H4MPT)
dehydrogenase.
 exact mechanism of catalysis under study, recent finding
suggests that molecular hydrogen is first heterolytically
cleaved by Fe(II), followed by transfer of hydride to the
carbocation of the acceptor.
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14. Fe only hydrogenase bonded with H2
 The Fe(II) electrophiles [Fe(dppe)2CN]+ ([1]+) and
[Fe(dppe)2CO]2+ ([2]2+) both form stable η2-H2
complexes, despite the contrasting
donor/acceptor properties of CN− and CO.
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16.  Biological hydrogen production
 various catalysts, either chemical or biological, can reduce
the produced H+ into H2.
 Hydrogenases are attractive since they require a relatively
low overpotential.
 its catalytic activity is more effective than platinum, which is
the best known catalyst for H2 evolution reaction.
 Low overpotential and high catalytic activity of [FeFe]
hydrogenases are accompanied by high O2 sensitivity.
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17.  Hydrogenase-based biofuel cells
 Typical enzymatic biofuel cells involve the usage of enzymes
as electrocatalysts at either both cathode and anode or at one
electrode.
 In hydrogenase-based biofuel cells, hydrogenase enzymes
are present at the anode for H2 oxidation.
 The generation of electricity from H2 is comparable with the
similar functionality of Platinum catalysts minus the catalyst
poisoning, and thus is very efficient.
 In the case of H2/O2 fuel cells, where the product is water,
there is no production of greenhouse gases.
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18.  The reversible reaction catalyzed by hydrogenase allows for the capture and
storage of renewable energy as fuel with use on demand.
 This can be demonstrated through the chemical storage of electricity
obtained from a renewable source (e.g. solar, wind, hydrothermal) as
H2 during periods of low energy demands.
 When energy is desired, H2 can be oxidized to produce electricity.
Advantages:
This is one solution to the challenge in the development of technologies for the
capture and storage of renewable energy as fuel with use on demand
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19. Hydrogenases have been investigated for a long time since
their discovery over 80 years ago.
However, significant problems involving O2-sensitivity and
complex maturation mechanisms have inhibited application
of hydrogenases and biological H2 production.
Hydrogenases have comparable catalytic activity to noble
metallic catalyst that humans invented, while they utilize
abundant metals on the earth such as nickel and iron.
They can operate under ambient condition with high
specificity.
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20.  Biological inorganic chemistry by Robert R.
Crichton
 David Schilter, James M. Camara, Mioy T. Huynh,
Sharon Hammes-Schiffer, and Thomas B.
Rauchfuss; Chem. Rev. 2016, 116, 8693−8749
 Chem rev. 2014,114,4081-4148
 Hydrogenase- wikipedia
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