α-Subunit oxidation in T-state crystals of a sebacyl cross-linked human hemoglobin with unusual autoxidation properties

In the crystal structure of human T-state hemoglobin with a sebacyl residue cross-linking the two β-subunit Lys⁸²'s (DecHb), the Fe atoms of the α-subunit hemes are found to be oxidized with a water molecule bound. The three-dimensional structure and heme geometries were compared to those of deoxyhemoglobin and other partially and fully oxidized hemoglobins [R. Liddington, Z. Derewenda, E. Dodson, R. Hubbard, G. Dodson, J. Mol. Biol. 228 (1992) 551]. The heme geometries of the α-subunits are consistent with those observed in oxidized structures. The proximal histidines of the α-subunits move toward the heme plane shifting the F-helix and FG-corner in a manner observed for partially oxidized human hemoglobin, This supports the hypothesis that these perturbations may precede the T- to R-state transition. Circular dichroism studies comparing DecHb and natural human hemoglobin in the deoxy and CO ligated forms confirm that the conformations of the deoxy forms are identical, but the ligated forms have slight differences in the solution structures. DecHb is found to be more resistant to autoxidation than natural hemoglobin. The time course of autoxidation of DecHb shows that it is virtually absent for the first 1500 min followed by a rapid increase. Thus, the discovery of the oxidation of the α-subunits in the deoxy-crystals is quite unexpected. The data confirm that ligation of the α-subunits precedes that of the β-subunits. This may suggest a low ligand affinity of the α-diligated form of hemoglobin.