The divalent cations Mg2+, Mn2+, Zn2+, Ca2+, and Ni2+ were found to protect against proteolysis a form of GroEL (ox-GroEL) prepared by exposing GroEL for 16 h to 6 mM hydrogen peroxide (H2O2). 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 H2O2). Ox-GroEL incubated with a divalent cation behaved like N-GroEL in that it could transiently interact with H2O2-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 H2O2, 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. (c) 2008 Elsevier Inc. All rights reserved.