Nickel-iron-sulfur clusters hydrogenases

Both hydrogenases and carbon monoxide oxidoreductases contain iron-sulfur clusters in addition to nickel. It may be noted that in addition to the Ni hydrogenases, there is another class of Fe hydrogenases, such as those in clostridia, which contain no nickel but have a specialized type of iron-sulfur cluster (28a, 28b). Therefore, it has to be established that the nickel in Ni hydrogenases is the active site as will be seen later, there is a considerable amount of circumstantial evidence for this. 

Fig. 7. Optical absorption and magnetic circular dichroism spectra of oxidized hydrogenase from M. thermoautotrophicum (AH strain), nickel concentration 120 pM. (a) Optical absorption spectrum, at room temperature the absorption is predominantly due to iron-sulfur clusters, (b) MCD spectra recorded at 1.53, 4.22, and 8.9 K, in a magnetic field of 4.5 T MCD is predominantly due to Ni(III), which is the only paramagnetic species in the oxidized enzyme. Reproduced, with permission, from Ref. 57.

Many of the Ni hydrogenases contain an iron-sulfur cluster presumed to be of the [3Fe-xS] type, which is paramagnetic with S = 1/2 in the oxidized state and S = 2 in the reduced state (68, 69). Thi function of these clusters is unknown. In some hydrogenases, typifie< by C. vinosum hydrogenase and the membrane-bound hydrogenase o Alcaligenes eutrophus (70), the EPR spectra of the iron-sulfur cluster and the oxidized nickel center show complex lineshapes (Fig. 8). In C vinosum the nickel is also EPR detectable and its spectrum also shows ... 

In D. gigas hydrogenase, splittings are not observed in the Ni-A and Ni-B signals from oxidized nickel centers (Fig. 4a), but are seen in the reduced Ni-C species at low temperatures (Fig. 5b) (41, 72). The splitting of Ni-C correlates with the reduced state of a [4Fe-4S] cluster (72). The spin-spin interactions observed in EPR are consistent with a distance between the nickel and iron-sulfur cluster of less than 1.2 nm (73). 

Fig. 10. Hypothetical reaction cycle for D. gigas hydrogenase, based on the EPR and redox properties of the nickel (Table II). Only the nickel center and one [4Fe-4S] cluster are shown. Step 1 enzyme, in the activated conformation and Ni(II) oxidation state, causes heterolytic cleavage of H2 to produce a Ni(II) hydride and a proton which might be associated with a ligand to the nickel or another base in the vicinity of the metal site. Step 2 intramolecular electron transfer to the iron-sulfur cluster produces a protonated Ni(I) site (giving the Ni-C signal). An alternative formulation of this species would be Ni(III) - H2. Step 3 reoxidation of the iron-sulfur cluster and release of a proton. Step 4 reoxidation of Ni and release of the other proton.

Each iron atom is terminally coordinated by one CO and one CN . The coordination of Fel is completed by the additional bridging cysteinyl sulfur. For Fe2, the sixth coordination site may be empty, as in the D. desulfuricans enzyme (Nicolet et al. 1999), or occupied by a solvent molecule, as observed in the C. pasteurianum enzyme (Peters et al. 1999). The assignment for the diatomic ligands is supported by infrared spectroscopic evidence (Pierik et al. 1998X and similar diatomic ligands have also been found for the corresponding binuclear [Ni-Fe] cluster of the nickel-iron hydrogenases (Volbeda et al. 1995). 

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