A Tour of Hydrogenase

The sulfate-reducing bacterium Desulfovibrio Gigas   [1]

The hydrogenases are the enzymes involved in the metabolism of dihydrogen. Through their activity, many microorganisms can use H₂ as an energy source (H₂ uptake) or use protons as an electron sink (H₂ evolution) according to the equation:

H₂     2H⁺ + 2e^-

There are three types of hydrogenases:

• The Nickel-Iron (NiFe) hydrogenases are both oxygen and light sensitive. They are usually involved in the H₂ uptake (for example in the methanogenic bacteria). This tour will focus on the NiFe hydrogenase from Desulfovibrio Gigas.;
• The Iron-iron (FeFe or Fe-only) hydrogenases are very air sensitive and are bi-directional but usually involved in the H₂ evolution (for example in Clostridia); and
• The Fe-hydrogenases that were considered to be metal-free until iron was discovered in 2000.

The Ni-Fe Hydrogenase

The wireframe display in the left screen shows the structure of hydrogenase form the sulfate-reducing bacterium Desulfovibrio Gigas. The enzyme has 800 amino acid residues and weighs 88 kDa.

  The secondary structure of the hydrogenase consists of 43 α-helices (red) and 16 β-sheets (blue).

  This display shows two subunits of hydrogenase. The small subunit (white) has 264 amino acid residues and weighs 28 kDa. The large subunit (blue) contains 536 amino acids and has a molecular weight of 60 kDa.

  The small subunit contains three iron-sulfur clusters: two 4Fe - 4S (labelled #1 and #2) and one 3Fe - 4S cluster. They lie in an almost straight line stretching from the surface of the protein to the subunit interface. The large subunit contains the enzyme's active site.

  The three iron clusters serve as electron carriers from a cytochrome protein to the active site. The 4Fe - 4S cluster, which is the closest to the surface of the enzyme (labelled -1) is an unusual Fe₄S₄ center. Commonly Fe₄S₄ clusters have 4 cysteine ligands while here one cysteine ligand is replaced by a histidine (His 185). This cys - his substitution probably gives the cluster a more positive redox potential. A Fe₃S₄ cluster is located in the middle while the second 4Fe - 4S cluster is in the proximity of the subunit interface and the active site.

The clusters are separated by approximately 6 Å.

This is the display of the active site and the 4Fe - 4S cluster closest to it. They are separated by approximately 6 Å.

This is a closer look at the oxidized active site. The active site is a binuclear, thiolate bridged Ni-Fe complex. The complex is coordinated by four cysteine ligands from the protein backbone. The nickel atom is coordinated by four cysteinate-sulfur atoms, of which two bridge to the iron atom. The identity of the third bridging species (the red sphere marked 'X' on the left structure) is unknown but it can be oxygen or hydroxyl ligand. Apart from the bridging sulfurs and the 'X' species, the iron atom is coordinated by three non-protein ligands: two cyano (CN^-) and one carbonyl (CO) ligand. The presence of these small ligands is quite surprising and without precedent in biological structures.

The bridging oxygen donor ligand X is lost when the active site is reduced. In the catalytic cycle, dihydrogen penetrates the protein, reaches the active site, and then binds at the iron(II) or nickel(II). Binding in an η², side-on fashion makes dihydrogen acidic. A proton is transmitted from dihydrogen to the protein surface via a network of hydrogen bonds. The loss of this proton leaves at the active site a hydride that bridges between the iron and nickel (i.e. X is a hydride now). There is the subsequent loss of electrons via the iron-sulfur cluster conduit to cytochrome electron carriers and the loss of a proton. This leaves Fe(II) and Ni(II) and a vacant site to bind another equivalent of dihydrogen.

The Fe-Fe Hydrogenase

This is the display of an Fe-Fe hydrogenase tracing the backbone of the protein.

  Like the NiFe hydrogenase, Fe-Fe hydrogenase has three FeS clusters to conduct electrons to/from the active site.

  Unlike the NiFe hydrogenases, the active site of FeFe hydrogenase is directly bonded to the nearest 4Fe-4S cluster via bridging cysteinate ligand. The ligands on the iron are thought to be (OC)(CN)Fe(SCH₂NHCH₂S)Fe(CO)(CN) where the azadithiolate ligand is represented in the crystal structure as a 1,3-propaneditholate ligand (the resolution is not good enough to distinguish between nitrogen and carbon.

  A series of protic side chains and water molecules provides a hydrogen-bonded network through which protons travel rapidly to and from the active site.

 Similarly there is a channel lined by hydrophobic sidechains that allows dihydrogen to access the active site.

  This is the structure of the active site. The "floating" oxygen atom actually belongs to a disordered carbonyl ligand (CO) that bridges between the two irons. Dihydrogen is thought to coordinate at the site trans to this carbonyl ligand.

The structure of the hydrogenase by Volbeda, A. et al. (1996) J. Am. Chem. Soc. 118(51), 12989-12996 (PDB id 2FRV).

If you would like to learn more about the hydrogenase and its model compounds see the following:

• Evans, D.J.; Pickett, C.J. (2003) Chem. Soc. Rev. 32, 268;
• Marr, A.C et al. (2001) Coord. Chem. Rev. 219-221, 1055;
• Darensbourg, M.Y. et al. (2000) Coord. Chem. Rev. 206-207, 533

Hydrogenase - Model Complexes

The development of the hydrogenase model complexes should provide important insights into the chemistry of this enzyme. However, the study of these synthetic analogues is not of purely scientific importance. A better understanding of hydrogenases might lead to better ways to produce dihydrogen for the "Hydrogen Economy", i.e. the utilization of dihydrogen as a clean energy source.

A Structural Model For the NiFe Active site

  The modeling of the NiFe hydrogenase active site is not an easy task. The structure on the left shows one of the proposed models. [2] The complex can be formulated as [(C₆H₄S₂)Ni (µ -'S'₃)Fe(CO)(PMe₃)₂] where 'S'₃ stands for the bis(2-mercaptophenyl)sulfide ligand. Like in the active site of the hydrogenase, the nickel atom is coordinated by four sulfur atoms two of which are bridging to the iron atom. However, the geometry around the Ni center is square planar rather than tetrahedral. The iron atom has a carbonyl and two PMe₃ ligands. The geometry around Fe is octahedral. Successful substitution of PMe₃ with cyano ligands has not been reported yet. This complex, despite its similarities with the hydrogenase's active site, does not react with the dihydrogen.

A Functional Model For the NiFe Active site

  Ogo et al. [3] report the synthesis, structure and reactivity of the bimetallic RuHNi complex shown at left. The complex can be formulated as [(C₆Me₆)Ru(µ-H)NiL]NO₃ where L stands for an S-N-N-S ligand N,N'-dimethyl-N,N'-bis(2-mercaptoethyl)-1,3-propanediamine. This complex has a hydride ligand and two thiolate ligands bridging between ruthenium(II) and nickel(II). Ruthenium is in the same group as iron. The active Ni-C state of hydrogenase has similar ligands bridging between Ni(III) and Fe(II). Like the Ni-C state, the Ru(H)Ni complex is formed by reaction of hydrogen gas with a bimetallic complex. It is a rare example of a paramagnetic transition metal hydride complex.

A Model For the FeFe Active site

  An interesting functional model is the red hydride complex (PMe₃)(CO)₂ Fe^II(µ-H)(SCH₂CH₂CH₂S)Fe^II(CO)₂(CN) with a hydride bridging between the two iron(II) ions. This complex is a catalyst for the electrochemical reduction of protons [4] .

[1]   Picture taken from Water Services Ltd.

[2]   Sellmann, D. et al. (2002) Angew. Chem. Int. Ed. 41(4), 632

[3]   Ogo, S. et al. (2007) Science 316, 585