Equilibrium dialysis of methionyl aminopeptidase from Escherichia coli (EcMetAP) monitored by atomic absorption spectrometry and magnetic circular dichroism (MCD) shows that the enzyme binds up to 1.1 +/- 0.1 equiv of Co2+ in the metal concentration range likely to be found in vivo. The dissociation constant, K-d, is estimated to be between 2.5 and 4.0 muM. Analysis of the temperature and magnetization behavior of the two major peaks in the MCD spectrum at 495 and 567 nm suggests that these transitions arise from Co2+ with different ground states. Ligand field calculations using AOMX are used to assign the 495 nm peak to Co2+ in the 6-coordinate binding site and the 567 nm peak to Co2+ in the 5-coordinate site. This is further supported by the fact that the binding affinity of the Co2+ associated with the 567 nm peak is enhanced when the pH is increased from 7.5 to 9.0, consistent with having an imidazole ligand from a histidine amino acid residue. On the basis of the MCD intensities, it is estimated that, when the 5-coordinate site is fully occupied, 0.1 equiv of cobalt is in the 6-coordinate site. Even when the cobalt concentration is very low, there is a small fraction of binuclear sites in EcMetAP formed through cooperative binding between the 5- and 6-coordinate Co2+ ions. The magnetization behavior of the 6-coordinate Co2+ MCD peak is consistent with an isolated pseudo-Kramer doublet ground state, suggesting that the cobalt ions in the binuclear sites are not magnetically coupled.