OXYGEN EQUILIBRIUM STUDIES OF CROSS-LINKED IRON-COBALT HYBRID HEMOGLOBINS - MODELS FOR PARTIALLY LIGATED INTERMEDIATES OF COBALT HEMOGLOBIN

To probe the molecular mechanism of allosteric function of cobaltous p rotoporphyrin-substituted hemoglobin (CoHb), a series of alpha, alpha- cross-linked symmetric and asymmetric Fe-Co hybrid hemoglobins, which contain (1Co porphyrin/3Fe porphyrins), (2Co porphyrins/2Fe porphyrins ), and (3Co porphyrins/1Fe porphyrin) per tetramer, have been prepared . Because only Fe porphyrin-containing subunits react with CO, these F e-Co hybrids are converted to mono-, di-, and tri-CO-ligated states in the presence of CO, respectively, and are proposed to stand as models for mono-, di-, and tri-ligated intermediates of CoHb, respectively. The oxygen binding properties of these Fe-Co hybrids were investigated by measuring oxygen binding isotherms in the presence of CO as a func tion of pH in the presence and absence of IHP. The ligation of CO to a beta subunit causes larger changes in the oxygen affinity and the Boh r effect than that to an alpha subunit, indicating that the ligation t o a beta subunit induces larger affinity-related structural changes in cross-linked CoHb. Di- and tri-CO-ligated intermediates exhibited sub stantially increased oxygen affinity, reduced Bohr effect, and reduced IHP effect, indicating that they are in high affinity states. Calcula tion of the Adair equilibrium constants for the first and last oxygena tion steps for each of these intermediates permitted the determination of the level of free energy of cooperation. The intermediately ligate d species of cross-linked CoHb are distributed in multiple levels of f ree energy of cooperation within the free energy difference of 1.14 kc al mol-1 between deoxy and fully ligated states of cross-linked CoHb a t pH 7.4. The ligation process in CoHb is determined by the number and distribution of the bound ligands, and ligation takes place through s teps that require minimal free energy changes.