• Prosthetic group features
• Reactions catalysed by hydrogenase
• Redox states of Ni-Fe hydrogenase
• The properties of the Ni-Fe centre
• The properties of the Fe-S clusters
• Ni-Fe hydrogenase in enzyme databases
• Ni-Fe hydrogenase in motif databases
• Ni-Fe hydrogenase in alignment databases
• Ni-Fe hydrogenase in 3­D databases
• References

Subunit	Prosthetic group	Formal oxidation states
Large	[Ni-X-Fe](SCys)4(CY)2(SO) X = S or O; Y = O or N	A (`unready, oxidised'): NiIII-FeII (S=½); B (`ready'): NiI-FeII (S=½); C (`active'): NiI-H¯-FeII (S=½); R (`fully reduced'): NiI-H¯-FeI or H¯-NiI-H¯-FeIII or H¯-NiII-H¯-FeII (S=0); SI (`active, oxidised'): NiI-FeI or NiI-H¯-FeIII or NiII-H¯-FeII (S=0); SU (`unready, partially reduced'): NiII-FeII (S=0)
	MgNHisOGluOLeu(H2O)3	MgII
Small	[Fe4S4](SCys)4	[Fe4S4]+; [Fe4S4]2+
	[Fe4S4](SCys)3NHis	[Fe4S4]+; [Fe4S4]2+
	[Fe3S4](SCys)3	[Fe3S4]0; [Fe3S4]+

Hydrogenases catalyse the reversible oxidation of molecular hydrogen (H₂) and play a vital role in anaerobic metabolism. Metal­containing hydrogenases are subdivided into three classes: Fe ("iron only") hydrogenases, Ni-Fe hydrogenases and Ni-Fe-Se hydrogenases [1]. Hydrogen oxidation is coupled to the reduction of electron acceptors such as oxygen, nitrate, sulphate, carbon dioxide and fumarate, whereas proton reduction (H₂ evolution) is essential in pyruvate fermentation or in the disposal of excess electrons.

The Ni-Fe hydrogenases, when isolated, are found to catalyse both H₂ evolution (1) and uptake (2), with low­potential multihaem cytochromes such as cytochrome c₃ acting as either electron donors D or acceptors A, depending on their oxidation state [2]:

2H+ + Dred H2 + Dox	(1)
H2 + Aox 2H+ + Ared	(2)

The EPR studies of Ni-Fe hydrogenases have shown the existence of three paramagnetic states called Ni­A, Ni­B and Ni­C; other forms of the hydrogenase called Ni­R, Ni­SI and Ni­SU are EPR­silent (see above; note that the actual oxidation states of the Ni and Fe in all the various intermediates remain to be determined experimentally). Forms R, C and SI participate in the catalytic cycle of the hydrogenase, while A, SU and B are involved in the activation and inactivation of the enzyme [3]:

B	e¯, H+ E0' = -150 mV	SI			e¯, H+ E0' = -380 mV	C	e¯, H+ E0' = -445 mV	R
		O2
		A	e¯, H+ E0' = -230 mV	SU

The 3­D structures of the Ni-Fe hydrogenases from Desulfovibrio gigas [4] and Desulfovibrio vulgaris [5] have been determined (see Figure 1FRV). The large subunit is an /ß protein. The active site is dinuclear, containing both Ni and Fe ions placed 2.55-2.9 Å apart. The Ni is pentacoordinated (square pyramidal) with four S atoms of Cys residues being equatorial ligands and the bridging S or O atom an axial ligand. The coordination geometry of the Fe is a slightly distorted octahedron, with three bridging ligands between Ni and Fe (two S of Cys residues and one S or O atom) and three terminal ligands called L1, L2 and L3 (Figure 1FRV b). In D. vulgaris hydrogenase, the larger ligand L1 has been proposed to be S=O, while the smaller ligands L2 and L3 have been assigned as CO or CN¯ [5]. Some ligand coordination properties of the Ni-Fe centre of the Ni-Fe hydrogenases from D. gigas and D. vulgaris are summarised in the following table (see corresponding references for details on active site distances and angles):

Species	Ni terminal ligands	Ni-Fe bridging ligands	Fe terminal ligands	Ref.
Desulfovibrio gigas	S (Cys­65, Cys­530)	S (Cys­68, Cys­533); O; (H¯)	L1, L2, L3 = CO, CN¯, ·NO, N2 or CCH¯	[6]
Desulfovibrio vulgaris Miyazaki	S (Cys­81, Cys­546)	S (Cys­84, Cys­549); S or O; (H¯)	L1 = SO; L2, L3 = CO or CN¯	[5]

There is no general agreement on the catalytic mechanism of Ni-Fe hydrogenase. Density functional theory (DFT) quantum chemical methods were used to probe the mechanism of H₂ activation by the enzyme [7]. In this proposed mechanism, the resting state of the dinuclear cluster is Ni^IIFe^III; H₂ first binds to Fe in the form of a molecular hydrogen complex, which then undergoes heterolytic splitting. Hydride transfer to Fe and proton transfer to the adjacent Cys thiolate ligand is accompanied by decoordination of the protonated Cys thiol from Ni while remaining bound to Fe. Simultaneously, the cyanide ligand on Fe binds with the Ni atom in a bridging binding mode. After the H₂ dissociation, the hydride bound to Fe can then be transferred to Ni which should be a necessary preliminary for subsequent H⁺ or electron transport.

In the C­terminus of the large subunit of D. vulgaris hydrogenase a Mg centre has been found, approximately 13 Å apart from the Ni-Fe centre. The Mg ion is octahedrally coordinated by the N of His­552, O² of Glu­62, the carbonyl oxygen of Leu­498 and three water molecules, Wat1, Wat2 and Wat3, which are further hydrogen bonded by O¹ and O² of Glu­337 and O¹ of Glu­62, respectively. The role of the Mg is unclear.

The small subunit consists of two domains, I_S and II_S. The /ß twisted open sheet structure of the N­terminal I_S domain is similar to that of flavodoxin; the C­terminal II_S domain contains two ­helices and no ß­structure. The Fe-S clusters are distributed almost along a straight line, with the [Fe₃S₄] cluster located half­way between the two [Fe₄S₄] clusters. The two [Fe₄S₄] clusters have been termed proximal (prox) and distal (dist) based on their distance to the Ni atom. Domain I_S binds the [Fe₄S₄]_prox cluster, whereas domain II_S binds [Fe₄S₄]_dist and [Fe₃S₄] clusters. The [Fe₄S₄]_dist cluster is coordinated by one His and three Cys residues. This is the only known example of histidine acting as a [Fe₄S₄] cluster ligand in protein structure. A crown of acidic residues surrounds the partially exposed His ligand of the [Fe₄S₄]_dist cluster and this might provide a recognition site for the redox partner (cytochrome c₃) [4]. The properties of the Fe-S clusters of the Ni-Fe hydrogenase from D. gigas are summarised in the following table:

Cluster	Redox potential (mV) [2]	Domain	Amino acid ligands
[Fe4S4]prox	-340	IS	Cys­17, Cys­20, Cys­112, Cys­148
[Fe4S4]dist	-290	IIS	His­185, Cys­188, Cys­213, Cys­219
[Fe3S4]	-35	IIS	Cys­228, Cys­246, Cys­249

A mechanism of electron transfer from the Ni active site through the Fe-S clusters to the cytochrome c₃ has been suggested [4]. The role of the [Fe₃S₄] cluster is not yet clear. The high redox potential of this cluster and its absence from some homologous hydrogenases put its involvement in electron transfer in doubt [4].

Nickel-iron hydrogenase in enzyme databases

ENZYME	LIGAND	BRENDA	Official name	Alternative name
1.12.2.1	1.12.2.1	1.12.2.1	Cytochrome­c3 hydrogenase	Hydrogenase
1.18.99.1	1.18.99.1	1.18.99.1	Hydrogenase	Hydrogenlyase

Nickel-iron hydrogenase in motif databases

PRINTS ID	PRINTS AC	PROSITE/BLOCKS ID	PROSITE AC	BLOCKS AC
-	-	NI_HGENASE_L_1 NI_HGENASE_L_2	PS00507 PS00508	BL00507
NIHGNASESMLL	PR00614	-	-	-

Nickel-iron hydrogenase in alignment databases

Protein Superfamily	Pfam	LPFC 3­D alignment
00175; hydrogenase (NiFe) large chain	PF00374; NiFeSe_Hases	-
00177; hydrogenase (NiFe) small chain	-	-

Nickel-iron hydrogenase in 3­D databases

PDB	MSD	scop	BSM	RELI Base	Header	¹
1frv	1frv	1frv	1frv	1frv	Nickel-iron hydrogenase (oxidised); Desulfovibrio gigas	MS6MB1
2frv	2frv	2frv	2frv	2frv	Nickel-iron hydrogenase (oxidised) (complex with Mg2+·3H2O); Desulfovibrio gigas	-

¹ Macromolecular Structures abstract. Full text is available to BioMedNet Members

References

1. Fauque, G., Peck, H.D., Jr., Moura, J.J.G., Huynh, B.­H., Berlier, Y., DerVartanian, D.V., Teixeira, M., Przybyla, A.E., Lespinat, P.A., Moura, I. and LeGall, J. (1988) The three classes of hydrogenases from sulfate­reducing bacteria of the genus Desulfovibrio. FEMS Microbiol. Rev. 4, 299-344.
2. Cammack, R., Fernandez, V.M. and Hatchikian, E.C. (1994) Nickel-iron hydrogenase. Methods Enzymol. 243, 43-68.
3. de Lacey, A.L., Hatchikian, E.C., Volbeda, A., Frey, M., Fontecilla­Camps, J.C. and Fernandez, V.M. (1997) Infrared­spectroelectrochemical characterization of the [NiFe] hydrogenase of Desulfovibrio gigas. J. Am. Chem. Soc. 119, 7181-7189.
4. Volbeda, A., Charon, M.H., Piras, C., Hatchikian, E.C., Frey, M. and Fontecilla­Camps, J.C. (1995) Crystal structure of the nickel-iron hydrogenase from Desulfovibrio gigas. Nature 373, 580-587.
5. Higuchi, Y., Yagi, T. and Yasouka, N. (1997) Unusual ligand structure in Ni-Fe active center and an additional Mg site in hydrogenase revealed by high resolution X­ray structure analysis. Structure 5, 1671-1680.
6. Volbeda, A., Garcin, E., Piras, C., de Lacey, A.L., Fernandez, V.M., Hatchikian, E.C., Frey, M. and Fontecilla­Camps, J.C. (1996) Structure of the [NiFe] hydrogenase active site: Evidence for biologically uncommon Fe ligands. J. Am. Chem. Soc. 118, 12989-12996.
7. Pavlov, M., Siegbahn, P.E.M., Blomberg, M.R.A. and Crabtree, R.H. (1998) Mechanism of H-H activation by nickel-iron hydrogenase. J. Am. Chem. Soc. 120, 548-555.

Bibliography on structural studies of nickel-iron hydrogenase

Reviews on nickel-iron hydrogenase