Studies on cobalt myoglobins and hemoglobins: XI. The interaction of carbon monoxide and oxygen with α and β subunits in iron-cobalt hybrid hemoglobins

Abstract

An artificial hybrid hemoglobin, α(Co) 2β(Fe) 2, the α and β subunits of which carry cobaltous protoporphyrin IX and ferrous protoporphyrin IX, respectively, and its complementary hybrid α(Fe) 2β(Co) 2 were prepared and the properties of their ferrous subunits were examined by equilibrium and kinetic measurements with carbon monoxide as a ligand. The β(Fe) 2 subunits in fully deoxy α(Co) 2β(Fe) 2 exhibited a higher affinity for carbon monoxide than did the α(Fe) 2 subunits in fully deoxy α(Fe) 2β(Co) 2. Addition of 2 m m-inositol hexaphosphate decreased fourfold the carbon monoxide affinity of the α(Fe) 2 subunits in α(Fe) 2β(Co) 2 and by more than tenfold that of the β(Fe) 2 subunits in α(Co) 2β(Fe) 2 at pH 7.0. The higher affinity for carbon monoxide of the β(Fe) 2 subunits inα(Co) 2β(Fe) 2 than that of the α(Fe) 2 subunits in α(Fe) 2β(Co) 2 was caused by a smaller dissociation rate of the β(Fe) 2-carbon monoxide complex. These results are considered to underlie the different affinity for carbon monoxide of the α and β subunits in deoxy hemoglobin. Oxygen equilibria of the cobaltous subunits in iron-cobalt hybrid hemoglobins were also measured in the presence of carbon monoxide. The α(Co) 2 subunits in α(Co) 2β(FeCO) 2 showed a higher oxygen affinity than the β(Co) 2 subunits in α(FeCO) 2β(Co) 2. Inositol hexaphosphate lowered the oxygen affinity of the β(Co) 2 subunits in α(FeCO) 2β(Co) 2 by eight-fold, but that of the α(Co) 2 subunits in α(Co) 2β(FeCO) 2¦by only 1.6-fold. The magnitude of the alkaline Bohr effect, as defined by Δlog P m Δ pH was found to be −0.34 and −0.14 for α(FeCO) 2β(Co) 2 and α(Co) 2β(FeCO) 2, respectively, in 0.1 m-phosphate buffer at 15 °C. The rate of oxygenation and deoxygenation of the cobaltous subunits in iron-cobalt hybrid hemoglobins in the presence of carbon monoxide was determined by a temperature-jump relaxation method in 0.1 m-phosphate buffer with and without inositol hexaphosphate. Their relaxation spectra were of a single exponential character and differed from that of cobalt hemoglobin. Without inositol hexaphosphate, the association rate constants for both α(Co) 2β(FeCO) 2 and α(FeCO) 2β(Co) 2 were close to that for cobalt hemoglobin, whereas the dissociation rate constants for iron-cobalt hybrid hemoglobins were smaller than that for cobalt hemoglobin by more than fourfold. Inositol hexaphosphate affected both the association and dissociation rates of α(FeCO) 2β(Co) 2 but did so to a lesser extent than those of α(Co) 2β(FeCO) 2. These observations suggest strongly a different role for the α and β subunits in the co-operative oxygenation and alkaline Bohr effect of hemoglobin.