Low-temperature optical spectroscopy of cobalt in Cu,Co superoxide dismutase: A structural dynamics study of the solvent-unaccessible metal site

The temperature dependence (300 to 10 K) of the electronic absorption spectra of the cobalt chromophore in bovine superoxide dismutase (SOD) having the native Zn(II) ion selectively replaced by Co(II) has been investigated in four different derivatives: Cu(II),Co(II) SOD, N-3(-)-Cu(II),Co(II) SOD, Cu(I),Co(II) SOD, and E,Co(II) SOD in which the copper ion has been selectively removed. In the Cu(II),Co(lI) SOD, the cobalt spectrum is characterized at room temperature by three bands centered at 18,472, 17,670, and 16,793 cm(-1); the low-frequency band is split, at low temperatures, into two components, indicating a lower symmetry contribution to a predominantly tetrahedral crystal field. Addition of N-3(-) to the Cu(II),Co(II) SOD introduces slight changes in all the Co(II) visible bands, indicating the occurrence of minor perturbations of the structural cobalt site upon anion binding to the catalytic copper site. Analysis of the spectra in the Cu(I),Co(II) and E,Co(II) enzymes indicates that the His61 imidazolate bridge is released from the copper upon reduction. This is also confirmed by the analysis of the zeroth, first, and second moments of the various bands in the different derivatives. The cobalt site is characterized by a harmonic dynamics, at variance with what observed in the solvent accessible copper site [Cupane, A., Leone, M., Militello, V., Stroppolo, M. E., Polticelli, F., & Desideri, A. (1994) Biochemistry 33, 15103-15109]. The degree of local microheterogeneity at the cobalt site is smaller than that observed for the copper site and increases in the order N-3(-)-Cu(II),Co(II)approximate to Cu(I),Co(II) < Cu(I),Co(II) < E,Co(II) indicating a different local packing and the presence of different constraints on the cobalt site in the four derivatives. The different dynamic behavior with respect to the catalytic, solvent-accessible, copper site is discussed.