Copper-substituted forms of the wild type and C42A variant of rubredoxin

In order to gain insights into the interplay between Cu(I) and Cu(II) in sulfur-rich protein environments, the first preparation and characterization of copper-substituted forms of the wild-type rubredoxin (Rd) from Desulfovibrio vulgaris Hildenborough are reported, as well as those of its variant C42A-Rd. The initial products appear to be tetrahedral Cu^I(S-Cys)_n species for the wild type (n = 4) and the variant C42A (n = 3, with an additional unidentified ligand). These species are unstable to aerial oxidation to products, whose properties are consistent with square planar Cu ^II(S-Cys)_n species. These Cu(II) intermediates are susceptible to auto-reduction by ligand S-Cys to produce stable Cu(I) final products. The original Cu(I) center in the wild-type system can be regenerated by reduction, suggesting that the active site can accommodate Cu ^I(S-Cys)₂ and Cys-S-S-Cys fragments in the final product. The absence of one S-Cys ligand prevents similar regeneration in the C42A-Rd system. These results emphasize the redox instability of Cu^II-(S-Cys) _n centers.