Ikeda-Saito M, Hori H, Inubushi T, Yonetani T
J Biol Chem. 1981 Oct 25;256(20):10267-71.
Cobalt-substituted leghemoglobin (CoLb) was prepared from cobaltous protoporphyrin IX and apo-leghemoglobin a, and its functional and spectroscopic properties were examined. The oxygen affinity of CoLb was pH-dependent. The partial oxygen pressure at half-saturation was decreased from 14.3 torr at pH 8 to 3.5 Torr at pH 3.5 with a pK value of 5.8 at 15 degrees C. The oxygen association and dissociation rates were determined by the temperature jump-relaxation technique. The association rate was essentially pH-independent, and the relatively high oxygen affinity of CoLb was found to be due to the fast oxygen association rate in comparison with cobalt myoglobin. The oxygen dissociation rate was pH-dependent with a pK value of 5.7 at 15 degrees C, suggesting that the pH dependence of the oxygen dissociation rate is responsible for the pH-dependent change in the oxygen affinity of CoLb. The EPR spectrum of oxyCoLb exhibited a pH-dependent change with a pK value of 5.7 at 15 degrees C. The EPR spectrum at 77 K of oxyCoLb at neutral pH exhibited a small effect upon the replacement of H2O with D2O, but it was drastically altered in the acidic pH region. The deoxy EPR spectrum was pH-independent between pH 4 and 9. These observations indicate that in oxyCoLb, the bound oxygen is interacting with the distal amino acid group with a pK value of 5.7 and that this interaction is responsible for the increased oxygen affinity in the acidic pH region.
