Isotropically shifted NMR resonances for the proximal histidyl imidazole NH protons in cobalt hemoglobin and iron-cobalt hybrid hemoglobins. Binding of the proximal histidine toward porphyrin metal ion in the intermediate state of cooperative ligand binding.
Exchangeable imidazole NH proton nuclear magnetic resonance (NMR) signals of the proximal histidine F8, which is directly coordinated to the paramagnetic porphyrin cobalt ion, have been measured for cobalt-substituted deoxyhemoglobins (deoxy-CoHb) and iron-cobalt hybrid hemoglobins, alpha (Co)2 beta (Fe)2 and alpha (Fe)2 beta (Co)2. Comparison of NMR spectra between these Co-substituted hemoglobins and natural Fe-containing hemoglobin allows the assignment of the NH signals to the specific subunits, namely, a signal at 53.8 ppm to the alpha-subunits and a signal at 58.4 ppm to the beta-subunits for deoxy-CoHb at 23 degrees C, respectively. The coordination of carbon monoxide to the beta-subunits in alpha (Co)2- beta (Fe)2 gave rise to an 8 ppm downfield shift of the proximal histidyl NH signal in the deoxy alpha (Co)2 subunits. In contrast, the coordination of carbon monoxide to the alpha-subunits in alpha (Fe)2 beta (Co)2 showed only a 1 ppm downfield shift for the deoxy beta (Co)2 subunits. These observations suggest that the metal-histidyl coordination bonds in the deoxy cobalt subunits are strengthened by the ligation of carbon monoxide in the counterpart ferrous subunits. However, the change of the coordination was much larger in the alpha (Co)2 subunits than that in the beta (Co)2 subunits. Line-width analysis of the NH signals for CO-bound hybrid hemoglobins gave a mean exchange lifetime of 3.2 X 10(3) S-1 for the T to R quaternary structural transition. The proximal histidyl binding situation at some liganded states is discussed in relation to the quaternary structure of hemoglobin molecules.