The ground and excited state properties of Co(lI) substituted for Zn(II) at the catalytic (c) and the noncatalytic (n) sites of horse liver alcohol dehydrogenase EE isozyme have been investigated by parallel EPR and UV/ visible variable-temperature magnetic circular dichroism (VTMCD) spectroscopies. Samples were investigated as prepared and after formation of a ternary complex with NAD(+) and the potent inhibitor pyrazole. In accord with the structural role proposed for the noncatalytic metal, the spectroscopic properties of Co(II) at the noncatalytic site were unperturbed by formation bf the ternary complex. The EPR spectra were readily analyzed in terms of a S = 3/2 spin Hamiltonian using anisotropic intrinsic g-values in the range characteristic of tetrahedral Co(II), i.e. g = 2.1-2.4; E/D approximate to 0.33, 0.05 (with D < 0), and 0 (with D > 0) for Co(c)Zn(n)-HLADH, Co(c)Zn(n)HLADH/NAD(+)/pyrazole, and Zn(c)Co(n)-HLADH, respectively. VTMCD studies facilitated resolution and assignment of S --> Co(II) charge transfer bands (300-400 nm) and the components of the (4)A(2) --> T-4(1)(P) tetrahedral d-d band (500-800 nm) that are split by spin-orbit coupling and low-symmetry distortions. The splittings of the highest energy d-d band are indicative of a much more distorted coordination environment for Co(II) at the catalytic site than the noncatalytic site This is also reflected in the magnitude of ground state zero-field splitting, Delta, determined by analysis of the temperature dependence of discrete MCD bands, Delta = 33, 56, and 7 cm(-1) for Co(c)Zn(n)-HLADH, Co(c)Zn(n)-HLADH/NAD(+)/pyrazole, and Zn(c)Co(n)-HLADH, respectively. MCD magnetization data are rationalized in terms of the EPR-determined ground state effective g-values, ground state zero-field splitting, and the polarization of the electronic transitions. The zero-field splittings for the samples with Co(II) at the catalytic site determined by VTMCD are quite different from those determined by EPR from the temperature dependence of the spin relaxation (Makinen, M. W.; Yim, M. B. Proc. Natl. Acad. Sci. U.S.A. 1981 78, 6221-6225), and the origin of this discrepancy is discussed. In accord with X-ray crystallographic studies, the EPR and VTMCD data are rationalized in terms of a highly distorted tetrahedral coordination environment for Co(II) at the catalytic site (two cysteines, one histidine, and one H2O for Co(c)Zn(n)-HLADH and two cysteines, one histidine and one pyrazole for Co(c)Zn(n)-HLADH/NAD(+)/pyrazole) and a more regular tetrahedral environment for Co(II) at the noncatalytic site (four cysteines).