A Volbeda

C Hatchikians

E Garcin

J C Fontecilla-Camps

M Frey

Y Montet

CiteSeerX

Pure & Appl. Chem., Vol. 70, No. 1, pp. 25-31, 1998. 
Printed in Great Britain. 
@ 1998 IUPAC 
Structural bases for the catalytic mechanism of 
NiFe hydrogenase 
Y. Montet*, E. Garcin*, A. Volbeda*, C. Hatchikians, M. Frey* and J.C. Fontecilla-Camps* 
* Laboratoire de Cristallographie et de Cristallogenkse des ProtBines, Institut de Biologie Structurale- 
Jean-Pierre-Ebel CEA-CNRS, 41 Avenue des Martyrs 38027 Grenoble - France 
0 Unit6 de BidnergCtique et IngCniCrie des ProtCines, CNRS, 31 Chemin Joseph Aiguier, 13402 
Marseille CDX 20 France. 
Introduction 
The reaction H2 c----> 2 H  + 2e' provides electrons at a significantly low potential (the H2/2H+ 
redox couple has a E? of -414 mV at pH 7 and 1 bar of H2). However, hydrogen oxidation is not 
energetically favourable because of the very low acidity of the H-H bond (pKa = 35). This can be 
dramatically changed through binding of molecular hydrogen to a metal center since the pKa can be 
lowered by as many as 20 to 30 units, (ref.l, 2). This, in turn, facilitates the heterolytic cleavage of the 
H2 molecule (giving H + Hi), a first step in the oxidation process. Hydrogen metabolism is present in 
a wide variety of chemotrophic and phototrophic microorganisms where hydrogen consumption or 
production are central features of such various processes as biogas production, denitrification, nitrogen 
fixation, corrosive sulfate reduction, production of H2 from water and specific hydrogenation reactions 
or cofactor regeneration (ref. 3,4). 
The enzymes responsible for H2 metabolism are called hydrogenases and have been classified 
as [NiFe] and pel-only enzymes (ref. 4). [NiFe] hydrogenases are heterodimeric with relatively well 
conserved 60kDa large subunits and more diverse small subunits (ref. 5 )  containing one or more 4Fe4S 
clusters (Fig. 1). It has been shown that hydrogen activation is mediated by the nickel center and that 
the iron-sulfur clusters are probably involved in electron transfer between the active site, the molecular 
surface and the redox partners of the enzyme, such as cytochromes and ferredoxins (ref. 5). 
EPR studies 
[NiFe] hydrogenases have been subjected to extensive EPR studies (see ref. 5 and references 
therein). Three paramagnetic Ni states, Ni-A, Ni-B and Ni-C, have been identified which depend on 0 2  
and the redox potential. Ni-A and Ni-B represent the EPR spectra produced by the most oxidized 
states. Neither of these forms seems to be part of the catalytic cycle which operates at significantly more 
negative potentials (ref. 6). The Ni-C signal corresponds to a partially reduced form with a bound 
hydrogen species supposed to be either H2, H' or H (ref. 5 ) .  The binding of a hydrogen species to the 
active site Ni center has been demonstrated by the isotopic effect induced by deuterium on the 
photolysis of Ni-C, as detected by EPR (ref. 7). Ni-C seems to be the only stable paramagnetic species 
which is part of the catalytic cycle. 
Redox titrations of D. gigas hydrogenase (ref. 6) have indicated that besides Ni-A/B and Ni-C, the 
active site can exist in two other EPR-silent states, NI-SI and Ni-R. The various redox states are 
separated by sequential one-electron reductions in the following order: Ni-B --> Ni-SI --> Ni-C --> Ni- 
25 
26 Y. MONTET eta/.  
Figure 1. Overall picture of the 
fold of the [NiFe] hydrogenase 
of Desulfovibrio gigas. 
Ribbons and arrows designate 
a-helices and P-strands, 
respectively. The large subunit 
is shown in dark grey, the 
small subunit in light gray. The 
latter binds one [3Fe-4S] and 
two [4Fe-4S] clusters. The 
color code for the cofactors is: 
black Fe, white Ni inorganic S .  
Redox titrations 
R. These and other studies (ref. 7) have led to the proposition of a cyclic catalytic mechanism involving 
Ni-SI (as the active H2 oxidant), Ni-C, and Ni-R (as the active H+ reductant). In addition to the active 
site redox center, D. gigas hydrogenase has two [4Fe-4S] clusters with midpoint redox potentials close 
to those of the Ni-SI/Ni-C and Ni-C/Ni-R couples, respectively. A full description of any overall state 
of this hydrogenase has to be expressed as a combination of the states of the active site and the two 
[4Fe-4S] clusters. This results in a complex combination of twelve possible microstates, forming three 
cycles, for which relative populations have been determined at various potentials (ref. 6).  
A simpler scheme, containing six microstates, can be proposed if only one [4Fe-4S] cluster is 
considered (which may be the case in some [NiFe] hydrogenases (ref. 5 ,  8). This minimal scheme 
contains two Ni-SI --> Ni-R --> Ni-C --> Ni-SI cycles (Fig. 2). Cycle I, which can only oxidize H2, 
seems to operate at potentials that are too positive for hydrogenase redox partner, the cytochrome c3 (E 
= -290 mV at pH 7). This suggests that either such cycle is not biologically relevant (except the S2 ---> 
R2 passage that provides access from cycle I to cycle 11), or that, at low P(H2), there may exist 
hydrogenase electron acceptors with a more positive redox potential than cytochrome c3. On the other 
hand, Cycle 11, operating at more reduced potentials, can both oxidize H2 and reduce protons. Upon 
H2 removal by flushing with an inert gas, two isoelectronic species (belonging to cycles I and 11) co- 
exist (ref. 6):  a Ni-C species with an oxidized FeS cluster and a Ni-SI state with a reduced one (C2 and 
S1, respectively, see Fig. 2). 
Figure 2. Simplified scheme, 
based on the work of Roberts 
and Lindahl (ref. 6) of 
ox a\ possible in a H2 "minimal" oxid tion [NiFe] cycl s
hydrogenase containing only 
one [4Fe-4S] cluster (the one 
proximal to the active site, see 
Fig. 1). S ,  C and R refer to 
subsequently more reduced 
states of the active site, 
whereas 1 and 2 label the 
oxidation state of the [4Fe- 
4S] cluster. Iso-electronic 
states are boxed. The external 
redox partner is denoted by 
ox and red. Only cycle I1 can 
operate in both directions. 
ox 
0 
Hf 
0 1998 IUPAC, Pure and Applied Cbernistry70.25-31 
Catalytic mechanism of NiFe hydrogenase 27 
These results have been discussed assuming that the active site of the [NiFe] hydrogenases was 
a mononuclear Ni center capable of performing both catalysis (H-H bond splitting) and redox chemistry 
(electron transfer) (ref. 5-7,9). There is little doubt that the Ni center constitutes the hydrogenase 
catalytic site, however, the assumption of Ni-based redox chemistry is more problematic. On the one 
hand, X-ray absorption (XAS) experiments have shown that the electron density at the Ni center does 
not change significantly as a function of the various redox states (ref. 10). On the other, the redox 
changes that the Ni center is supposed to undergo, namely Ni(II1) (Ni-AIB) -->Ni(II) (Ni-SI) -->Ni(I) 
(Ni-C) --->Ni(O) (Ni-R), (ref. 5) are difficult to reconcile with the results from model chemistry which 
indicate that, for instance, the Ni(III)/Ni(II) and Ni(II)/Ni(I) redox couples are too far apart to be 
accomodated within the range of observed redox potentials in hydrogenases (ref. 11 ). 
Crystallographic results and IR spectroscopy. 
Our X-ray crystallographic analysis of D. gigas [NiFe] hydrogenase has revealed that the active 
site is, in fact, binuclear with a second metal center, that we have shown to be iron, located at less than 
3 8, from the nickel ion (figs. 1 and 3 and ref. 12, 13). The Ni ion binds only four protein ligands. 
Cysteines 65 and 530 are terminal ligands whereas cysteines 68 and 533 bridge the two metals centers. 
We have also shown that the Fe center binds three non-protein diatomic ligands (Fig. 3 and ref. 13). 
The discovery of such an unexpected Ni-Fe center has deep implications for the understanding of 
biological H2 catalysis and requires a re-interpretation of the relevant available data. For instance, the 
existence of a binuclear center provides an alternative to a Ni ion which would be both catalytic and 
extensively redox active. Since the second metal site is occupied by a Fe ion, then at least some of the 
redox chemistry could take place at this site. Indeed, recent studies on the FI’IR analyses of 
Chromatium vinosum and D. gigas hydrogenases (ref. 13, 14) have shown the presence of three 
intrinsic high frequency IR bands normally absent from protein spectra. Furthermore, it was observed 
that the three bands shifted, in a concerted fashion, as a function of the redox state of the enzyme. It is 
almost certain that the IR bands ion, which have now been assigned to one CO and two CN-’s (ref. 
15), arise from the three non-protein ligands of the active site Fe. Consequently, they report on redox 
changes being sensed by this center. 
0 
Figure 3. The heterobinuclear 
active site of D .  gigas 
hydrogenase. The Fe center 
has three ligands: one CO and 
two CN”s. The Ni is ligated 
by four thiolates, two of 
which also bind to the Fe. 
The question mark designates 
a ligand (probably oxygen) 
that bridges the two metal 
centers in the oxidized 
enzyme. The Ni coordination 
es distorted square pyramidal, 
the empty axial site is trans to 
the Sy of Cys 533. 
0 1998 IUPAC, Pure and Applied Chemistry70,25-31 
28 Y. MONTET eta/.  
Gas accessibility to the active site. 
In order to locate possible H2 transfer pathways between the active site and the molecular 
surface, a cavity map was calculated from the model of D. gigas hydrogenase using a probe radius of 
1.0 A (Fig. 4A). Since all observed ordered solvent molecules were included in the model, only 
internal hydrophobic cavities at least 2.0 8, in diameter were selected. The result is a network of 
hydrophobic channels whose shape roughly resembles the letters V and A connected through their right 
and left sides, respectively (Fig. 4A). A channel starting at the interface between the small and the large 
subunit of hydrogenase extends from the bottom of the V motif to the protein surface. The left arm of 
the V is surrounded entirely by residues of the large subunit and shows several invaginations pointing 
towards the bulk solvent. The right leg of the A, which is located in the N-terminal domain of the small 
subunit, provides another possible access for gases to the active site. From this analysis, it appears that 
C 
Figure 4: A.  Cavity map of D. 
gigas hydrogenase calculated 
using a 1.0 A probe. B.  
Experimental xenon sites deduced 
from the peak maxima are shown 
as spheres, the electron density 
map is depicted as a grid. C. A 
close-up view of the cavity region 
near the active site. The cavity 
peters out at about 4.5 8, from the 
Ni and 4.0 8, from the Sy of Cys 
530. The clusters are designated 
A, B and C. 
0 1998 IUPAC, Pure and Applied Chemistry70,25-31 
Catalytic mechanism of NiFe hydrogenase 29 
there are several putative entrance sites for gas diffusion connected by different channels to a unique 
entrance to the active site. 
To probe gas accessibility to these hydrophobic channels, xenon binding experiments were 
carried out using crystals of the Ni-Fe hydrogenase of Desulfovibrio fructosovorans , which shows 
65% amino acid sequence identity with the D. gigas enzyme (ref. 16). Given the hydrophobic nature 
and high atomic number of Xe, its binding is a convenient way to investigate the gas accessibility of 
protein interiors by crystallographic methods (refs. 17,18). X-ray diffraction data for the Xe derivative 
of Desulfovibrio fructosovorans hydrogenase was prepared using a high-pressure capillary set-up. Ten 
major peaks were found (Fig. 4B), showing heights ranging from 4.1 to 8.3 times the root mean 
square (rms) value of the map. The protein environment of each of the Xe sites is highly hydrophobic 
and 85% of the amino acid residues within 4.5 8, from these sites are identical in D. gigas and D. 
fructosovorans hydrogenases. 
The end of the hydrophobic channel that approaches the active site is about 4.5 8, from the Ni 
ion and points at its empty axial coordination site, Cys 530 (that is substituted by a selenocysteine in 
some hydrogenases; ref. 19) is also close to this channel and could play the role of a base in the 
heterolytic cleavage of molecular hydrogen (Fig. 4C). Thus, our cavity analysis provides indirect 
support for Ni being the primary H2 binding site through its vacant axial coordination site. 
Figure 5. Possible H2 
oxidation cycles 
(marked I and II) in D. 
putative oxygen species 
observed in the crystal 
occupies the position of 
a hydride in the Ni-C 
state (see text). States 
shown at the same level 
are both isoelecaonic 
H+ and isoprotonic. For 
simplicity, only the 
active site metal ions 
and those hydrogen 
species that may be 
directly involved in 
catalysis are shown. 
Model Chemistry 
The active site of [NiFe] hydrogenases has been extensively modelled (for a recent review see 
ref. 20) Of primary importance are those compounds that can shed light on the influence of the 
coordination sphere and valence state of the Ni ion on the catalytic properties of hydrogenases. Studies 
by the groups of Holm (1 l), Darensbourg (21) and Mascharak (1) have highlighted the importance of 
S-ligands in the coordination sphere of the Ni ion. Both sulfur coordination and a Ni(1) redox state 
seem to be requirements for H2 activation and subsequent hydride formation. Although these studies 
have focused on mononuclear Ni compounds, their results concerning hydrogen binding and activation 
should apply to the binuclear active site of hydrogenases since the Ni ion is very likely to constitute the 
catalytic site in these enzymes. 
0 1998 IUPAC, Pure and Applied Chemistry70,25-31 
30 Y. MONTET eta/ .  
Catalytic mechanism 
From the recent data on the structure and function of [NiFe] hydrogenase it is possible to 
propose two plausible schemes for hydrogen biocatalysis. Based on the minimal scheme proposed 
above (fig. 2) the process can be divided into 4 different steps. 
1) H2 binding : Several lines of evidence suggest that the primary binding site of molecular hydrogen is 
the Ni center. At relatively high potentials, the most likely candidate would be a Ni-SI state (ref. 6) .  
However, if this is a Ni(1) species (see above), then the two metals at the active site must be coupled in 
order to generate a diamagnetic center. A likely state for Ni-SI would then be Ni(1)-Fe(1). At more 
reduced potentials, the H2 binding species could be Ni-C with an oxidized FeS cluster (ref. 9). Since 
the Ni-C state is one electron more reduced than the Ni-SI state and paramagnetic, a plausible electronic 
state for this species is Ni(1)-Fe(II) with a bound hydride. We have suggested that H- could bridge the 
two metal centers (ref. 12). Substrate binding and heterolytic cleavage by a Ni-C species would form a 
double hydride (fig. 5) .  This is possible since the Ni coordination in the putative Ni-C form wolud be 
distorted square pyramidal and, consequently, there would be an available ligand site. H2 (followed by 
H ) binding to the vacant site would make the Ni coordination transiently octahedral (see 3) below). 
2)  H2 cleavage : From the hydrogenase active site geometry and our Xe binding and H2 diffusion 
simulations (see above) it can be assumed that, in any case, H2 binds to the vacant axial coordination 
site (Fig. 3). If this is the case, the Cys530 thiolate is a good candidate for the base that would bind the 
proton resulting after heterolytic cleavage, whereas the newly formed hydride would have different 
fates depending on which is the binding Ni species. 
3) H- binding : If the primary oxidant is Ni-SI (ref. 6) then the hydride would move to a bridging 
position and the resulting Ni-R species (Fig. 5 )  could be Ni(1)-Fe(1) with a bridging hydride. If, on 
other hand, the oxidant is a Ni-C state with one oxidized FeS cluster, a transient species would form, 
three electrons more reduced than Ni-SI. This species would have a bridging hydride and a terminal 
one bound to the primary substrate site and a Fe(I1)-Ni(1) metal redox state. This, or an isoelectronic 
species, would then give an electron to the FeS cluster to form the Ni-R state. 
4) Redox cycling : In both cases, the transfer of one electron from the Ni-R active site to a FeS cluster 
would (re)generate the Ni-C state. The cycle will be closed either by the subsequent one-electron 
oxidation of Ni-C to give Ni-SI or by oxidation of the FeS cluster of the Ni-C species (electron transfer 
towards the redox partner). In scheme 1, the Ni ion may stay as Ni(1) throughout catalysis whereas the 
Fe ion shuttles between Fe(1) (Ni-SI and Ni-R) and Fe(I1) (Ni-C). In scheme 2, the Ni-R could be a 
double hydride Ni(I1)-Fe(I1) species. The two schemes are not mutually exclusive and one or the other 
may operate preferentially depending on the physiological conditions. 
Conclusions 
Recent structural and functional results have furthered our understanding of H2 biocatalysis at 
the molecular level. The presence of a heterobinuclear center in hydrogenases suggests that the catalytic 
and redox sites may be different. The Ni ion appears as the most likely catalytic site, given the general 
0 1998 IUPAC, Pure and Applied Chemistry 70,25-31 
Catalytic mechanism of NiFe hydrogenase 31 
characteristics of this metal. In turn, redox activity, as indicated by IR spectroscopy, may be mostly 
carried out by the Fe center. 
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0 1998 IUPAC, Pure and Applied Chemistry 70,25-31 


Structural bases for the catalytic mechanism of [NiFe] hydrogenases

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Structural bases for the catalytic mechanism of [NiFe] hydrogenases

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