The molecular mechanism of autoxidation for human oxyhemoglobin. Tilting of the distal histidine causes nonequivalent oxidation in the beta chain.

Human oxyhemoglobin showed a biphasic autoxidation curve containing two rate constants, i.e. kf for the fast autoxidation due to the alpha chains, and ks for the slow autoxidation of the beta chains, respectively. Consequently, the autoxidation of the HbO2 tetramer produces two different curves from the pH dependence of kf and ks. The analysis of these curves revealed that the beta chain of the HbO2 tetramer does not exhibit any proton-catalyzed autoxidation, unlike the alpha chain, where a proton-catalyzed process involving the distal histidine residue can play a dominant role in the autoxidation rate. When the alpha and beta chains were separated from the HbO2 tetramer, however, each chain was oxidized much more rapidly than in the tetrameric parent. Moreover, the separated beta chain was recovered completely to strong acid catalysis in its autoxidation rate. These new findings lead us to conclude that the formation of the alpha1beta1 contact produces in the beta chain a conformational constraint whereby the distal histidine at position 63 is tilted away slightly from the bound dioxygen, preventing the proton-catalyzed displacement of O-2 by a solvent water molecule. The beta chains have thus acquired a delayed autoxidation in the HbO2 tetramer.