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 cop per ion has been selectively removed. In the Cu(II),Co(lI) SOD, the co balt spectrum is characterized at room temperature by three bands cent ered at 18,472, 17,670, and 16,793 cm(-1); the low-frequency band is s plit, at low temperatures, into two components, indicating a lower sym metry contribution to a predominantly tetrahedral crystal field. Addit ion of N-3(-) to the Cu(II),Co(II) SOD introduces slight changes in al l the Co(II) visible bands, indicating the occurrence of minor perturb ations of the structural cobalt site upon anion binding to the catalyt ic copper site. Analysis of the spectra in the Cu(I),Co(II) and E,Co(I I) enzymes indicates that the His61 imidazolate bridge is released fro m the copper upon reduction. This is also confirmed by the analysis of the zeroth, first, and second moments of the various bands in the dif ferent derivatives. The cobalt site is characterized by a harmonic dyn amics, at variance with what observed in the solvent accessible copper site [Cupane, A., Leone, M., Militello, V., Stroppolo, M. E., Poltice lli, F., & Desideri, A. (1994) Biochemistry 33, 15103-15109]. The degr ee 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) indicatin g a different local packing and the presence of different constraints on the cobalt site in the four derivatives. The different dynamic beha vior with respect to the catalytic, solvent-accessible, copper site is discussed.