2
production is known in green algae (e.g. Chlamydomonas, Scenedesmus and Chlorella)
(Vignais 2007, Chader 2009), in protozoans (e.g. Trichomonas vaginalis), in anaerobic
ciliates (e.g. Nyctotherus ovalis) and in anaerobic fungi (e.g. Neocallimastix frontalis); the
latter three microorganisms possess hydrogenosomes, organelles dedicated to hydrogen
metabolism (Vignais 2007).
In any case the key components of hydrogen metabolism are two classes of enzymes:
hydrogenases and nitrogenases.
Hydrogenases are a large family of enzymes that are both able to produce and consume
hydrogen, as it will be described in details in paragraph 1.3. Hydrogen production in
Clostridia (Vignais 2007), in some cyanobacteria (Dutta 2005) and in green algae (Melis
2004) is due to hydrogenases.
Nitrogenase is the enzymatic complex that is responsible for atmospheric nitrogen gas
fixation into ammonia, so that it is available for other metabolic pathways. The reaction
has not a defined stoichiometry and can be written as (Rees 2005):
N2 + (6+2n)H+ + (6+2n)e- + p(6+2n)ATP 2NH3 + nH2 + p(6+2n)ADP + p(6+2n)Pi
Nitrogen fixation leads also to production of hydrogen gas. Moreover, nitrogenases are
reported to be active also in the absence of nitrogen and in this case they only catalyze
hydrogen production (Koku 2002, Vignais 2007). Nitrogenases are highly oxygen sensitive,
being rapidly inactivated. Hydrogen production in PNS bacteria (Koku 2002) and in some
cyanobacteria (Dutta 2005) is due to nitrogenases.
In the last years, biological hydrogen production has being investigated with different
approaches as a possible source of hydrogen gas in a future fossil fuels-free economy
(Rupprecht 2006).
An important feature of biological hydrogen production is that it may be coupled to waste
disposal (Kapdan 2006), because different kinds of wastes can be used as a substrate for
the growth of Clostridia or PNS bacteria.
More pioneering studies have been carried out on the direct conversion of solar energy into
hydrogen thanks to the coupling of photosynthesis and hydrogen production in the same
cell: in PNS bacteria, cyanobacteria and green algae (Esper 2006, Melis 2006, Ghirardi
2007, Beer 2009).
The possibility of practical applications of these processes is under study at various levels
(e.g. culture conditions, strains selection, metabolic and genetic engineering) and is facing
various issues (Vignais 2007, Beer 2009).
The engineering and use of hydrogenases and other biological components in artificial
devices, which are inspired to what nature has evolved for billion of years, is emerging as a
3
promising powerful technology (Esper 2006, Cracknell 2008, Hambourger 2008, Lubitz
2008, Reisner 2009).
Moreover, hydrogenases are considered good models for future catalysts that are not based
on expensive and poorly available noble metals (De Lacey 2007, Sbraccia 2008,
Armstrong 2009).
Since hydrogenases have a key role in biological hydrogen production, their study is of
great interest to improve biological hydrogen production and to ease its future practical
applications.
1.2 The nano-leaf
The construction of artificial devices based on biological components or their mimics that
are able to convert solar energy into hydrogen has been envisaged by many (Lubitz 2008).
The development of such a device is the main goal of the “SOLHYDROMICS” project,
supported by the European Commission’s 7th Framework Programme.
The device (fig. 1.1) is a sort of “nano-leaf” because it is inspired to some features of
photosynthesis; it will be essentially composed of two electrodes separated by a particular
membrane. The anode will carry an immobilized photosystem II (or a chemical mimic) that
will capture solar light and use the energy to split water into molecular oxygen, protons
and low potential electrons. Electrons and protons are then transferred through the
membrane to the cathode, carrying an immobilized hydrogenase (or a chemical mimic)
then will produce hydrogen. In such a device light capture, water hydrolysis into hydrogen
and oxygen should be self- sustained.
The design and assembly of the “nano-leaf” is a complex work that involves many
scientific aspects and technological challenges. The availability and characterization of a
Figure 1.1. Schematic representation of the “nano-leaf”, an artificial device under
development that should be able to directly convert solar energy into hydrogen gas.
4
highly active hydrogenase is of exceptional interest for the development of such a device
within the “SOLHYDROMICS” project.
1.3 Hydrogenases
Hydrogenases are a family of redox enzymes that are central in hydrogen metabolism in
many microorganisms, both prokaryotic and eukaryotic. They are able to catalyze the
reversible reaction H2 2H+ + 2e- in both directions (Fontecilla-Camps 2007, Vignais
2007). The direction of the reaction depends on the redox potential of hydrogenase’s redox
partners: in the presence of H2 and an electron acceptor (high potential), hydrogen will be
oxidized, while in the presence of an electron donor (low potential) hydrogen will be
produced.
The number of physiological redox partners of hydrogenases is large: NAD(P)+/NAD(P)H,
cytochromes, coenzyme F420, ferredoxins. The first hydrogenase classification was based
on the identity of the cofactor, and still today the EC classification is based on it (Vignais
2007).
Today hydrogenases are more properly classified in three classes on the basis of the metal
content and organization of the active site (fig. 1.2), since this difference is associated with
phylogenetically distinct sequences (Vignais 2007). The three classes are:
• [FeFe]-hydrogenases (for historic reasons they are also called “iron-only”
hydrogenases): the active site (named “H-cluster” or HC) is composed by a peculiar
[2Fe] subcluster coordinated by several non-protein ligands and bridged to a [4Fe4S]
subcluster by the thiol of a cystein (Peters 1998, Fontecilla-Camps 2007).
Hydrogenases belonging to this class are usually monomeric, but also dimeric, trimeric
and tetrameric forms exist; in any case these enzymes are highly modular, being
Figure 1.2. Schematic structure of the active site of the three classes of hydrogenases (modified from
Vignais 2007 and Shima 2008).
5
composed by the catalytic domain and by some other accessory domains containing
FeS centres (Meyer 2007, Vignais 2007). [FeFe] hydrogenases are widely distributed
in Bacteria and in some Eukaryotes (Meyer 2007).
• [NiFe]-hydrogenases: the active site is composed by a binuclear centre made of an
iron atom and a nickel atom coordinated by four cysteines and by non-protein ligands.
Also [NiFe]-hydrogenases have a modular structure; they are at least heterodimeric,
being at least composed by a large subunit (having the catalytic activity) and a small
subunit involved in electron exchange since it contains up to three redox active FeS
centres (Fontecilla-Camps 2007); other accessory subunits can be found (Vignais
2007). [NiFe] hydrogenases are found in Archaea and Bacteria.
[NiFeSe] hydrogenases are a subclass of [NiFe] hydrogenases, having similar structure
(Fontecilla-Camps 2007), but with the peculiarity that one of the cysteines
coordinating the nickel atom is substituted by a genetically encoded selenocysteine
(Garcin 1999).
• [Fe]-hydrogenases (for historic reasons they are also called “metal-free” or “iron-
sulfur-cluster-free”): this class is the smallest and it is found only in some
hydrogenotrophic methanogenic Archaea. The reaction catalyzed by these enzymes is
particular and is the reversible reduction of methenyltetrahydromethanopterin
(methenyl-H4MPT+) with H2 to methylene-H4MPT and H+. The active site is a
mononuclear iron centre coordinated to a guanylyl pyridone cofactor that can be
extracted from the enzyme by denaturation. These enzymes are homodimeric (Shima
2008).
Today about 450 hydrogenase sequences are known (Vignais 2007) and for each class
several crystal structures exist. As an example, the PDB ID of some solved structures are
listed below.
• [FeFe] hydrogenases: 1FEH (Peters 1998), 1HFE (Nicolet 1999), 3C8Y (Pandey
2008);
• [NiFe] hydrogenases: 1FRV (Volbeda 1995), 1H2A (Higuchi 1997);
• [Fe] hydrogenases: 3F47 (Shima 2008).
A common feature to all hydrogenases is a higher or lower sensitivity to oxygen (Vignais
2007, Meyer 2007).
6
1.4 [FeFe]-hydrogenases
Whereas [NiFe]-hydrogenases mainly catalyze H2 oxidation, [FeFe]-hydrogenases are
usually involved in hydrogen production under physiological conditions, with some
exceptions in both classes (Vignais 2007).
[FeFe]-hydrogenases are very sensitive to molecular oxygen, most of them being
irreversibly inactivated by this molecule (Vignais 2007). Indeed they are mostly found in
obligate anaerobic bacteria, such as those of the genus Clostridium, Desulfovibrio,
Thermotoga, and some purple bacteria (Meyer 2007).
Interesting exceptions are represented by the [FeFe] hydrogenases found in some
eukaryotes: green algae and protists. In green algae, such as Chlamydomonas reinhardtii
(Ghirardi 2007), hydrogenases are nuclear encoded, but the active protein is found in the
chloroplast stroma and their expression is highly regulated and activated only during
anaerobiosis (Forestier 2003). Other algal hydrogenases were identified in Scenedesmus
obliquus (Florin 2001), Chlorella fusca (Winkler 2002), Chlamydomonas moewusii and
Chlorococcum submarinum (Kamp 2008).
In protists, such as Trichomonas vaginalis, [FeFe]-hydrogenases are found in particular
organelles named hydrogenosomes (Meyer 2007, Vignais 2007) and are involved in ATP
production, giving H2 as product.
Interestingly, genes with high sequence homology were also identified in Saccharomyces
cerevisiae, Oryza sativa, Drosophila melanogaster and Homo sapiens. In this case the
hydrogenase activity is lost (Balk 2004, Huang 2007, Song 2008), and the gene evolved to
new functions, such as iron-sulphur proteins assembly (Balk 2004, Song 2008) or hypoxia
response (Huang 2007).
1.4.1 Structural features
[FeFe] hydrogenases display very high modularity (fig. 1.3, Vignais 2007, Meyer 2007),
and are at least composed by the H domain, containing the active site H-cluster, and
eventually by other accessory domains. Within the H-cluster domain, three signature
sequences were identified (Vignais 2007); they contain the four cysteines that coordinate
the prosthetic active site (fig. 1.4).
The smallest known [FeFe] hydrogenase is found in green algae (fig. 1.3: M1, ~45 kDa),
and is just composed by the H domain, containing the active site. In this case the redox
partners of the enzyme directly interact with the catalytic domain. These enzymes are very
interesting because they accept low potential electrons from the photosynthetic electron
transfer chain (Ghirardi 2007).
7
A very small hydrogenase-like protein
(16 kDa) was identified in Enterobacter
cloacae (Mishra 2004); it is formed by
different parts of the H domain, but it
lacks important residues, it is not
demonstrated to be a complete gene and
it has a very low hydrogenase activity.
For this reasons it is not fully considered
a hydrogenase (Meyer 2007).
The accessory domains appear in the
hydrogenases from Megasphaera
elsdenii (fig. 1.2: M2, ~50 kDa),
Desulfovibrio desulfuricans and D.
vulgaris Hildenborough (fig. 1.3:
D(M2)); the latter two enzymes are
heterodimeric because a part of the H
domain is on a different polypeptide.
This accessory domain possesses two
[4Fe4S] clusters coordinated by 8
cysteines organized
in a particular
sequence, and it is
homologous to a
class of ferredoxins.
In Clostridia, such as
C. pasteurianum
(pdb 1FEH) and C.
acetobutylicum, [FeFe] hydrogenases (fig. 1.3: M3, ~65 kDa) possess three accessory
domains. The first accessory domain from the N-terminal contains a [2Fe2S] centre
coordinated by four cysteines and it is homologous to plant ferredoxins, while the second
contains a [4Fe4S] coordinated by three cysteines and a histidine; the third is the same
described above in M2 enzymes. Other Clostridia possess a particular domain with eight
conserved cysteines (fig. 1.3: M3a).
A domain homologous to NuoE and thioredoxin-like [2Fe2S] ferredoxin and another one
homologous to NuoF are found in bigger hydrogenases. These domains are located on
Figure 1.3. Scheme of [FeFe]-hydrogenases modular
structure (modified from Vignais 2001 and Meyer 2007).
The domains are not to scale.
Figure 1.4. Signature sequences of [FeFe]-hydrogenases (from Vignais 2007).
Sequences are in PROSITE format: brackets include the residues occurring at a
single position, “x” means any residue and bold residues are fully conserved. The
edges were numbered according to the C. pasteurianum sequence. The active site
coordinating cysteines were highlighted in green.
Name Pattern
FeFe_P1 296 [FILT][ST][SCM]C[CS]P[AGSMIV][FWY] 303
FeFe_P2 352 [FILV][MGTV]PCxxK[DKQRS]x[EV] 361
FeFe_P3 495 ExMxCxxGCxxG[AGP] 507
8
different polypeptides: in Thermotoga maritima the hydrogenase is trimeric (fig. 1.3:
TR(M4)), in Desulfovibrio fructovorans and Thermoanaerobacter tengcongensis it is
tetrameric (fig. 1.3: TE(M3)) and in Nyctotherus ovalis it is monomeric (fig. 1.3: M5, ~120
kDa), containing all the described domains on the same polypeptide.
1.4.2 Active site structure
The knowledge about the active site
of [FeFe] hydrogenases was obtained
from studies of enzymes from
Desulfovibrio and Clostridium
species (Fontecilla-Camps 2007).
The H-cluster, that is the active site
of [FeFe] hydrogenases (fig. 1.5), is
composed by a [2Fe] subcluster
bridged to a standard [4Fe4S]
subcluster by a thiol of cysteine 503
(residue numbers are referred to C. pasteurianum hydrogenase, CpI), that is the last
cysteine in the signature sequence FeFe_P3 (fig. 1.4).The [2Fe] subcluster is composed by
two iron atoms (Fe1 or Fep for proximal and Fe2 or Fed for distal) that are coordinated by
two CN ligands, three CO ligands (one them bridging the two iron atoms), a dithiolate
ligand and a molecule of water. The [2Fe] subcluster is linked to the protein only by the
thiol of cysteine 503 that also coordinates the [4Fe4S] subcluster. The latter is also
coordinated by cysteines 300, 355 and 499.
Assignment of the identity of the non-protein ligands of the H-cluster was not simple, as in
crystal structures usually they cannot be univocally identified; spectroscopic studies were
useful (Pierik 1998, Fontecilla-Camps 2007). Recently, a refinement of the crystal
structure (Pandey 2008) led to the precise assignment of the CO and CN ligands, on the
basis of the slightly different length of the coordinating bonds.
Another important issue is the chemical identity of the γ-atom of the dithiolate bridging the
two iron atoms. It is usually assigned to be a nitrogen atom (Fontecilla-Camps 2007,
Vignais 2007, Silakov 2009a), but it was also proposed to be an oxygen atom (Pandey
2008). Since different studies were done on enzymes from different microorganisms with
different techniques, it is still to be clearly demonstrated if the identity of this atom is the
same in the whole class of [FeFe] hydrogenases.
In either case, this atom may have an important role in proton exchange between C299,
Figure 1.5. H-Cluster, structure of the active site of
Clostridium pasteurianum CpI [FeFe]-hydrogenase (from
Pandey 2008).
9
which is thought to be the first step for proton pathways from the surface, and distal Fe2,
where catalysis is carried out (Peters 1998, Meyer 2007, Fontecilla-Camps 2007, Pandey
2008).
The role for the [4Fe4S] subcluster is to carry low potential electrons from the accessory
FeS clusters to the [2Fe] subcluster, which is the real responsible for hydrogenase activity.
The H-cluster must not be considered as the sum of two kind of FeS clusters, but as an
electronically inseparable [6Fe] cluster due to extensive delocalization of frontier
molecular orbitals (Schwab 2006). This is interesting in algal hydrogenases, where the H-
cluster is the only prosthetic group present.
Although the kinetics of the reaction are too rapid to be directly studied, some redox states
of [FeFe]-hydrogenases were identified
(fig. 1.6) by EPR, FTIR and Mössbauer
spectroscopy (reviewed in De Lacey
2007). Hinact (also named Hoxair) is the
inactive oxidized state; Hinact form of
some hydrogenases (e.g those from
Desulfovibrio vulgaris and D.
desulfuricans) can be converted in the
active Hox form under reduction in
anaerobiosis, through the intermediate
Htrans. The Hox form is most probably that
observed in the crystal structures
(Fontecilla-Camps 2007). The active Hred
form can be obtained by one electron
reduction of Hox and this causes a shift of
the bridging CO ligand toward distal Fe2. At very low potentials, an instable super-reduced
state Hsred was identified. The CO-inhibited form was identified as a Hox form with the
bound CO.
CO inhibition of [FeFe]-hydrogenases is known as a competitive process (De Lacey 2007);
the binding site of CO was identified by X-ray crystallography as the distal Fe2 atom of the
active site (Lemon 1999), and confirmed by spectroscopy data.
Figure 1.6. Redox states of [FeFe]-hydrogenases (from
De Lacey 2007).
10
1.4.3 Catalysis
The catalytic cycle for hydrogen production implies the following steps (Peters 1999, De
Lacey 2007):
• intermolecular electron transfer from a low potential electron partner to the accessory
FeS clusters;
• intramolecular electron transfer from the accessory FeS clusters to the active site;
• proton transfer from the surface to the active site, through protonable residues and
structural water molecules;
• formation of diatomic hydrogen at the active site;
• diffusion of hydrogen out of the molecule.
Hydrogen oxidation is thought to follow the same pathway in the opposite direction.
Hydrogen oxidation was demonstrated to be a heterolytic cleavage, thus leading to a single
proton and a hydride intermediate that is then oxidized to a proton. The site for this
reaction is the distal Fe2 atom (Peters 1999, De Lacey 1007); the observation that he
competitive inhibitor CO binds in this position (Lemon 1999) further confirms this model.
In the very proximity of the active site, proton transfer may be mediated by the amine of
the dithiolate bridging ligand (or by the oxygen atom) and by the very close cysteine (C299
in CpI).
On the basis of these knowledge, slightly different models have been proposed (De Lacey
2007), also by DFT calculations (Bruschi 2008, Sbraccia 2008). The two forms involved
are Hox and Hred. Electrons are transferred one by one to the [2Fe] sublcluster; distal Fe2 is
Figure 1.7. A possible catalytic cycle of [FeFe]-hydrogenases (from De Lacey
2007). X=CH2NHCH2.
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