Magnetic circular dichroism of DCPIP-oxidised Desulfovibrio vulgaris hydrogenase

Ni-free hydrogenases commonly exhibit a g av > 2 EPR spectrum in their most oxidised level. This spectrum has been linked with the active site and generally attributed to an oxidised HIPIP-type, [4Fe-4S] 3+ cluster. We report the low temperature magnetic circular dichroism (MCD) spectrum of 2,6-dichlorophenolindo-phenol (DCPIP)- oxidised Desulfovibrio vulgaris hydrogenase (DvH 2ase), which exhibits an axial g av > 2 EPR spectrum. Paramagnetic MCD is observed, which however is shown to arise from an EPR-silent, para-magnetic species with S > 1 2 . No evidence of paramagnetic MCD arising from the g av > 2 EPR-detectable species is obtained. We conclude that: (1) DCPIP-oxidised DvH 2ase does not contain either a [4Fe-4S] 3+ or an oxidised 3Fe cluster; (ii) an EPR-silent species with MCD characteristics somewhat similar to those of the oxidised ‘P’ clusters of the dye-oxidized iron-molybdenum protein of nitrogenase is present in DCPIP-oxidised DvH 2ase; (iii) the MCD and EPR of the g av > 2 species exhibit characteristics in common with those of the g av > 2 species produced by Fe(CN) 6 3− oxidation of the [4Fe-4S] 2+ cluster of the 7Fe ferredoxin I of Azotobacter vinelandii [(7Fe)FdI). We suggest that the g av > 2 EPR signal of this and other hydrogenases arises from a species chemically analogous to that observed in (7Fe)FdI.