Co(II) derivatives of Cu,Zn-superoxide dismutase with the cobalt bound in the place of copper: A new spectroscopic tool for the study of the active site

Abstract

Co(II) derivatives of Cu,Zn-superoxide dismutase having cobalt substituted for the copper (Co,Zn-superoxide dismutase and Co,Co-superoxide dismutase) were studied by optical and EPR spectroscopy. EPR and electronic absorption spectra of Co,Zn-superoxide dismutase are sensitive to solvent perturbation, and in particular to the presence of phosphate. This behaviour suggests that cobalt in Co,Zn-superoxide dismutase is open to solvent access, at variance with the Co(II) of the Cu,Co-superoxide dismutase, which is substitued for the Zn. Phosphate binding as monitored by optical titration is dependent on pH with an apparent p K a = 8.2. The absorption spectrum of Co,Zn-superoxide dismutase in water has three weak bands in the visible region ( ε = 75 M −1·cm −1 at 456 nm; ε = 90 M −1·cm −1 at 520 nm; ε = 70 M −1·cm −1 at 600 nm) and three bands in the near infrared region, at 790 nm ( ε = 18 M −1·cm −1), 916 nm ( ε = 27 M −1·cm −1) and 1045 nm ( ε = 25 M −1·cm −1). This spectrum is indicative of five-coordinate geometry. In the presence of phosphate, three bands are still present in the visible region but they have higher intensity ( ε = 225 M −1·cm −1 at 544 nm; ε = 315 M −1·cm −1 at 575 nm; ε = 330 M −1·cm −1 at 603 nm), whilst the lowest wavelength band in the near infrared region is at much lower energy, 1060 nm ( ε = 44 M −1·cm −1). The latter property suggests a tetrahedral coordination around the Co(II) centre. Addition of 1 equivalent of CN − gives rise to a stable Co(II) low-spin intermediate, which is characterized by an EPR spectrum with a highly rhombic line shape. Formation of this CN − complex was found to require more cyanide equivalents in the case of the phosphate adduct, suggesting that binding of phosphate may inhibit binding of other anions. Titration of the Co,Co-derivative with CN 2 provided evidence for magnetic interaction between the two metal centres. These results substantiate the contention that Co(II) can replace the copper of Cu,Zn-superoxide dismutase in a way that reproduces the properties of the native copper-binding site.