Hydrogenases:
Electrocatalysis and
Implications for Future Energy
Technology
Fraser Armstrong
Department of Chemistry
Oxford
H2(g) + O2(g)  H2O (liq)
DH = -286 kJ/mol
specific enthalpy -143 kJ/gram H2
NASA uses hydrogen fuel
to launch the space shuttles.
Sunday Telegraph
December 21 2003
The scientific challenges for a
future H2-based energy economy
synthesis
from fossil fuels
from solar
physical
(electrolysis and
photocells)
storage
chemical,
cryo,
compression
biological
(fermentative
and photosynthetic)
energy
conversion
combustion
fuel cell
Pt catalyst
enzyme, organism
or ‘smart’ catalyst
(bioinspired)
Chemical and Engineering News
July 22, 2002
Volume 80, Number 29
pp. 35-39
RENEWABLE Hydrogen bubbles form inside flask of
sulfur-deprived green algae. PHOTO BY MICHAEL
BARNES, UNIVERSITY OF CALIFORNIA
Juan Fontecilla-Camps
Fe-only hydrogenase
NiFe-hydrogenase
Active site of [NiFe]-hydrogenase
The active site of
hydrogenases con
center coordinate
invariant Cys thi
which are termin
An additional
O-ligand is present
in inactive states
O
The other two Cy
bridge the Ni and
centers. The latte
coordinated by o
two CNs.
In the oxidized fo
a putative oxo lig
coordination of F
octahedral and t
square pyramida
Identified by crystallography and FTIR
Chemical and Engineering News
July 22, 2002
Volume 80, Number 29
CENEAR 80 29 pp. 35-39
ISSN 0009-2347
CAN WE EXPLOIT
HYDROGENASES?
Insights into nature's substitute
for platinum may lead to the
design of inexpensive catalysts
MICHAEL
FREEMANTLE, C&EN
LONDON
ACTIVE SITE Oxford University Ph.D. student Sophie E. Lamle
investigates activity of hydrogenase docked on an electrode.
PHOTO BY MICHAEL FREEMANTLE
Fuel cells can be as small as we like, even micro
The future....fuel cells with cheap, inexpensive specific
electrocatalysts, perhaps without a membrane ?
Ideas from Nature
ANODE
Hydrogenase
H2
Ni-Fe
O2
CATHODE
high E oxidases ?
oxidase
electrons
Power ?
On Pt, H2 is cleaved homolytically,
Industry has to cope with CO
H ........H
H-
H+
Pt
M
base
Pt
At an enzyme, H2 is cleaved heterolytically. Spectroscopic
studies reveal a Ni(III)-hydrido species as an intermediate
(Ni is formally oxidised by H2)
+
H
Cys65
H
S
Ni III
Cys530
S
CN
-
CN
Fe
S
S
CO
Cys68
Cys 533
Structure of
[NiFe]-hydrogenase
from Desulfovibrio gigas
H2
H+
Fe
Ni
-Subunit
(contains
the active
site)
-Subunit
(contains
the electron
relay)
[4Fe-4S]dist
[3Fe-4S]
[4Fe-4S]prox
Other [NiFe]-hydrogenases have similar sequences
or spectroscopic properties
What we expect from chemistry alone
O2 + 4H+ + 4e-
2H+ + 2e-
2H2O
1.23 V
(0.82)pH7
H2
0.00 V
(-0.41)pH7
Based on flat electrode, no hydrodynamic assistance
maximum current for H2 oxidation is about 4 mA/cm2 at ambient
temperature and 1 bar gas (from diffusion coefficient of H2).
Similar figures for O2.
Power expectation is then about
whatever the catalyst is
4 mW/cm2,
Our mission..
How good are hydrogenases for
Electrocatalysis ?
How do they behave on an electrode ?
What are the challenges for their
widespread technological application?
Investigating hydrogenases by protein film voltammetry
H2
H+
hydrogenase
electrode
surface
Measure
catalytic current
= turnover rate
Control chemistry by
modulating electrode potential
Protein Film Voltammetry: Catalytic action can produce
a large current with characteristic dependence on potential
Normalised
current
e-
1
O
R
Potential/ Volts
0
-0.3
0
0.3
-1
-2
-3
-4
product
substrate
Current = Turnover rate
At steady-state,
rate is function of
potential, not time
-5
-6
Guides to interpretation:
Heering et al. J. Phys. Chem, B 102, 6889 (1998)
Léger et al. Biochemistry 40, 11234 (2001).
Léger et al. J. Phys. Chem. B. 106, 13058 (2002).
Léger et al. Biochemistry 42, 8653 (2003).
Enzyme is adsorbed on rotating disc Pyrolytic Graphite ‘Edge’
electrode. Less than 10 femtomole of enzyme is addressed, and
numerous consecutive experiments can be conducted on same sample.
enzyme and
co-adsorbate
(polyamine)
H
O
H3N+
HO
H
NH3+
H
H
H
NH3+
H O
OH O
+
H3 N
H
H
H3N+
H
H
HO
Special rotating-disc cell for studying gas enzymes
designed by Kylie Vincent
potential
controller / data
collection
electrode
rotator –
up to
3500
rpm
saturated
calomel
reference
electrode
o-ring seal onto
electrode rotator
gas out
septum for
injection of
liquids
gas in
water
out
Pt wire counter electrode
water in
a few picomoles of
protein adsorbed on
spinning graphite
electrode surface
water jacket for
temperature control
Preparing the film: Stationary PGE electrode is potential-cycled
in dilute H2ase solution ( < 1 mM) (in this case D.gigas NiFe enzyme)
1.5
Re-oxidation of H2
produced by H+ reduction
1.0
i / mA
0.5
0.0
-0.5
H+ reduction
-1.0
-1.5
-0.6
-0.4
-0.2
E / V vs SHE
0
0.2
Catalytic voltammograms for film of NiFe hydrogenase
adsorbed at a rotating disc PGE electrode under 0.1 atm H2.
(Pershad et al Biochemistry 38, 8992 (1999)
Based on detectable coverage limit, kcat >> 1500 sec-1 at 30 oC
2x
[4Fe-4S]2+/+
+/0
[3Fe-4S]
3800 rpm
15.0
3000 rpm
Current / mA
2000 rpm
1000 rpm
5.0
E (H+/H2)
i
-0.4
-0.2
keeps increasing
as rotation rate is
increased.
lim
Buffer
-5.0
-0.6
H2 oxidation rate
0
Potential vs SHE
0.2
Scan rate 0.2 V/s
Voltammograms for electrocatalytic Hydrogen oxidation
Pt/PGE 100% H2
H2ase/PGE 100% H2
4
3
2
1
4
3
2
1
0
-0.4
-0.05
c)
Current Density /(mA cm-2)
b)
Current Density /(mA cm-2)
Current Density /(mA cm-2)
a)
H2ase/PGE 10% H2
0.5
0.4
0.3
0.2
0.1
0
0.3
Potential /V vs. SHE
-0.4
-0.05
0
0.3
Potential /V vs. SHE
-0.4
-0.05
0.3
Potential /V vs. SHE
(Jones et al. Chem. Commun. 866 2002).
Levich plots for H2 oxidation by [NiFe]-H2ase and Pt catalyst
bound at identical or same-sized electrodes
(Jones et al. Chem. Commun. 866 2002).
Turnover rate estimated in region of 10,000 sec-1 at 45 oC
 NiFe
hydrogenase on PGE
 Pt on PGE
D Pt on Au
4
3
2
1
0
0
30
60
rotation rate1/2 /(rpm 1/2)
1 % Hydrogen
Current Density /(mA cm-2)
10 % Hydrogen
Current Density /(mA cm-2)
Current Density /(mA cm-2)
100 % Hydrogen
0.6
0.4
0.2
0
0
30
60
rotation rate1/2 /(rpm 1/2)
0.06
0.04
0.02
0
0
30
60
rotation rate1/2 /(rpm 1/2)
solution
adsorbed H2ase molecules
graphite electrode
4
3
Platinum on Gold
2
1
0
0
200
400
600
Tim e /s
Current Density /(mA cm-2)
Inhibition by CO is
easily reversed
at [NiFe]-H2ase
but not Pt
Current Density /(mA cm-2)
CO
CO
3
2
AvH2ase
1
0
0
200
Time /s
400
‘100%- Bio’ hydrogen fuel cell : no chemical catalysts
H2ase (NiFe
enzyme) on
PGE
electrode
laccase
(Cu enzyme)
on PGE
electrode
O
2
Nafion
membrane
Power (micro
Watts)
H2
Max power output
80
60
40
20
0
-1
-20
1
3
5
log[R] (R in k ohm)
7
We have established....
•Active site of Allochromatium vinosum [NiFe]-hydrogenase
can oxidise H2 at diffusion controlled rate, comparable to
Platinum catalyst, over wide pH range.
•CO inhibition is easily reversible
(and can be avoided completely, see later)
Challenges.
•Need to control and stabilise enzyme adsorption.
(Experiments with new porous carbon materials)
•Hydrogenases inactivated by oxidants.
•For all future applications of hydrogenases, we need to
understand the mechanisms of inactivation and activation
and, with collaboration from other experts, find solutions
to these problems.
Hydrogenases operate at low potentials
Microorganisms have to cope with O2
concentration
surface 0
5m
10 m
15 m
20 m
max
O2
aerobic
bacteriochlorophyll
anaerobic
sulfide
sediment
Oxidation inactivates NiFe hydrogenases:
a bridging ligand X blocks the site
NiIII
INACTIVE (OXIDISED)
CN
Cys65
X
S
Ni III
Cys530
S
S
CO
S
oxidants
O2
?
CN
Fe
Cys68
Cys
ACTIVE (REDUCED)
533
H2 ?
NiII
CN
Cys65
S
CN
Ni
Cys530
S
Fe
S
S
Cys
CO
Cys68
533
reductants