The divalent cations Mg(2+), Mn(2+), Zn(2+), Ca(2+), and Ni(2+) were found to protect against proteolysis a form of GroEL (ox-GroEL) prepared by exposing GroEL for 16h to 6mM hydrogen peroxide (H(2)O(2)). K(+) and other monovalent cations did not have any effect. Divalent cations also induced a conformational change of ox-GroEL that led to the decrease of its large exposed hydrophobic surfaces (exposed with H(2)O(2)). Ox-GroEL incubated with a divalent cation behaved like N-GroEL in that it could transiently interact with H(2)O(2)-inactivated rhodanese (ox-rhodanese), whereas ox-GroEL alone could strongly interact with ox-rhodanese. Although, ox-GroEL incubated with a divalent cation could not recover the ATPase activity (66%) lost with H(2)O(2), it could facilitate the reactivation of ox-rhodanese (>86% of active rhodanese recovered), without requiring ATP or the co-chaperonin, GroES. This is the first report to demonstrate a role for the divalent cations on the structure and function of ox-GroEL.