Electron paramagnetic resonance studies on cobalt hemoglobin, iron-cobalt hybrid hemoglobins, and their related model complexes. Characterization of proximal histidine binding to porphyrin cobalt(II) ion and its transition associated with subunit interaction.

Abstract

Electron paramagnetic resonance studies have been conducted on iron-cobalt hybrid deoxyhemoglobins and cobalt deoxyhemoglobin. Examination of alpha (Co)2 beta (Fe)2 hybrid hemoglobin at two different microwave frequencies revealed two sets of axial-symmetric electron paramagnetic resonance (EPR) signals rather than the anisotropic splitting of the g signal as reported previously [Ikeda-Saito, M., Yamamoto, H., & Yonetani, T. (1977) J. Biol. Chem. 252, 8639-8644]. One of these two sets is a characteristically broad spectrum with g = 2.38 and g parallel = 2.03; the other, a sharp spectrum with g = 2.33 and g parallel = 2.03. The alpha-subunits of deoxy-CoHb also exhibited a complex spectrum with a mixture of these two different types of signals. The relative compositions of these two signals were calculated for deoxy-alpha (Co)2 beta (Fe)2 and the alpha-subunits of CoHb, which were profoundly sensitive to the pH of the medium and also to the presence of allosteric effectors. A positive correlation was noticed between the spectral characteristics and the equilibrium extent of oxygenation, showing that the broad component may be associated with the electronic state of the porphyrin metal ion in T-state hemoglobin and the sharp component with that in the R state. In contrast, the complementary hybrid alpha (Fe)2 beta (Co)2 and the beta-subunits in CoHb showed relatively narrow spectra under similar conditions, and only small changes were noted with variation of pH and addition of inositol hexaphosphate. These spectra were close to the sharp component of the alpha-subunits associated with the R state. Comparison of the hemoglobin EPR spectra with those of model cobaltous porphyrin complexes revealed that the restraint that is operative at the fifth ligand position of the prosthetic group in the T state is released in the R state. These ligand binding properties were discussed in relation to the regulation of oxygen binding to hemoglobin.