| Abstract: | An artificial hybrid hemoglobin, alpha(Co)2 beta(Fe)2, the alpha- and beta-subunits of which contain cobaltous and ferrous protoporphyrins IX, respectively, and its complementary hybrid, alpha(Fe)2 beta(Co)2, were prepared from human hemoglobin, crystallized in the deoxy state, and examined by electron paramagnetic resonance (EPR) spectroscopy. The orientations of the porphyrin normals in these deoxy Fe-Co hybrid hemoglobins in terms of the g parallel signals, were closely coincident with those of the heme normals of deoxyhemoglobin determined by x-ray crystallography. Two sets of axially symmetric EPR signals were found in the alpha(Co)-subunits, whereas only one set was observed in the beta(Co)-subunits. Nonequivalence in the electronic structures of the prosthetic groups between the two alpha(Co)-subunits, designated alpha I and alpha II, within deoxy-alpha(Co)2 beta(Fe)2 hybrid hemoglobin was correlated to these two distinct EPR signals. The interaction between the epsilon-nitrogen of the imidazole ring of the proximal histidine and the cobaltous ion in deoxy-alpha I(Co)-subunit is different from that in the deoxy-alpha II(Co)-subunit. The absence of a strict molecular dyad axis in the deoxy-alpha(Co)2 beta(Fe)2 hybrid hemoglobin suggests that the affinity state of the alpha(Co)-subunits may be partially switched to the R-state having a higher affinity for oxygen. Upon partial ligation of carbon monoxide to the beta(Fe)-subunits, the line width of the g parallel and perpendicular signals of the alpha II(Co)-subunit was found to become somewhat narrower without disruption of the crystal structure. This suggests that there may be very close contacts between the alpha- and beta-subunits of different hemoglobin molecules which appear to be responsible for stabilizing the deoxy crystal structure after partial ligation in the crystalline state. |
