A Co(II)-substituted form of the R2 protein of ribonucleotide reductase from Escherichia coli has been prepared by anaerobic addition of 4 equiv of Co(II) to the R2apo protein in order to explore structural changes that may occur when divalent metal ions are coordinated in the metal binding sites. The visible absorption and circular dichroism spectra of the Co(II)-substituted R2 protein contain multiple absorption features in the 500-600-nm region with two apparent maxima at 515 and 550 nm (epsilon550 = 115 M-1 cm-1/Co(II)) indicative of Co(II) in a five-coordinate environment consisting of nitrogen or oxygen ligands. The intensity of the g = 5.44 signal in the EPR spectrum of the Co(II)-R2 complex, typical of S = 3/2 high-spin mononuclear Co(II), indicates that the Co(II) moieties are uncoupled when bound to the R2 protein. The efficient electronic relaxation properties of Co(II) in this coordination environment have enabled us to obtain remarkably high-resolution paramagnetically shifted H-1 NMR spectra, relative to the diferrous and diferric protein, despite the 87 kDa size of R2. Preliminary assignments of several of these resonances are made. The spectroscopic properties described here remain unchanged when the protein is eluted from an air-saturated gel filtration column suggesting that, unlike the very air-sensitive diferrous R2 protein, the Co(II)-R2 complex does not readily bind or activate dioxygen.