Studies on Cobalt Myoglobins and Hemoglobins

Artificial myoglobins and hemoglobins containing cobaltous proto-, meso-, and deuteroporphyrins (proto-, meso-, and deutero-CoMb and -CoHb) have been prepared reproducibly by an anaerobic recombination of apomyoglobin from sperm whale and apohemoglobin from human blood with cobaltous porphyrins in the presence of pyridine. These CoMb and CoHb contain one cobalt porphyrin per heme binding site and are capable of reversible oxygenation, confirming the original observation of Hoffman and Petering ((1970) Proc. Nat. Acad. Sci. U. S. A. 67, 637–643). Their optical absorption characteristics and extinction coefficients have been established. By means of the automatic oxygenation measurements of Imai et al. ((1970) Biochim. Biophys. Acta 200, 189–196), the following values have been obtained for the partial pressure of oxygen in torr at half-saturation (p0.5) and Hill's coefficient (n) at half-saturation. The p0.5 values of CoMb are 50 to 100 times larger than those of the corresponding iron-containing myoglobins (FeMb) and are constant in a range from pH 6.5 to 8.0 in both the presence and absence of inositol hexaphosphate. The p0.5 values of CoHb are 10 to 25 times larger than the corresponding iron-containing hemoglobins (FeHb). The alkaline Bohr effects (∂ log p0.5/∂ pH) for proto-, meso-, and deutero-CoHb are 0.5, 0.3, and 0.3, respectively. The p0.5 value of CoHb increases 3-fold upon addition of 500 µm inositol hexaphosphate in 0.1 m phosphate buffer, pH 7.0. The effect of chemical modifications of porphyrin side chains on the oxygen affinity of iron- and cobalt-containing oxygen carriers has been found to vary considerably depending on the nature of apoprotein and temperature. The observed sequence of p0.5 is: proto- g meso- g deuteroporphyrins for CoMb and FeMb above 25°, meso- g proto- g deuteroporphyrins for cobaltous porphyrin dimethylesters, CoMb, and FeMb below 20°, and proto- g deutero- g mesoporphyrins for CoHb and FeHb at 3–30°. These variations of the p0.5 sequence suggest that the effect of side chain modifications on the oxygen affinity is stereochemical rather than electronic. Protoporphyrin IX is required for a minimal oxygen affinity and a maximal cooperativity in CoHb. Thermochemical comparison of oxygenation parameters of iron- and cobalt-containing oxygen carriers indicates that the effect of the metal substitution on the oxygen affinity is primarily enthalpic, whereas the effect of the apoprotein binding on the oxygen affinity of cobaltous porphyrins is essentially entropic.