Cobalt Bovine Superoxide Dismutase. Reactivity of the Cobalt Chromophore in the Copper-Containing and in the Copper-Free Enzyme

Abstract

The reactivity of the Zn site of bovine superoxide dismutase [EC 1.15.1.1] was probed by observing optical and EPR changes, under several conditions, of the Co(II)-substituted protein. Only in the absence of Cu are the optical and EPR spectra of the Co chromophore appreciably affected by alkaline pH or by cyanide. With both reagents the reaction with the Cu-containing protein appears to involve the water molecule bound to the Cu and does not affect the magnetic coupling between Cu and Co. The reaction of cyanide with the Cu-free Co(II) protein leads to a slow detachment of Co from the protein as pentacyanocobalt. An oxygen adduct forms in air, analogous to that described in Co(II)carbonic anhydrase [EC 4.2.1.1.]. Acid titration modifies the Co(II) spectra in the same way in the Cu-containing and in the Cu-free protein and brings about uncoupling of the Co(II)-Cu(II) system. Protonation of histidine-61 on the zinc facing nitrogen is suggested. H2O2 modifies the Co chromophore only in the presence of Cu. Magnetic coupling between Cu(II) and Co(II) seems to be still present after H2O2 inactivation of the enzyme.