Effect of Heme Modification on Oxygen Affinity of Myoglobin and Equilibrium of the Acid--Alkaline Transition in Metmyoglobin.

Functional regulation of myoglobin (Mb) is thought to be achieved through the heme environment furnished by nearby amino acid residues, and subtle tuning of the intrinsic heme Fe reactivity. We have performed substitution of strongly electron-withdrawing perfluoromethyt (CF3) group(s) as heme side chain(s) of Mb to obtain large alterations of the heme electronic structure in order to elucidate the relationship between the O2 affinity of Mb and the electronic properties of heme peripheral side chains. We have utilized the equilibrium constant (pKa) of the "acid-alkaline transition" in metmyoglobin in order to quantitatively assess the effects of the CF3 substitutions for the electron density of heme Fe atom (ρFe) of the protein. The pKa value of the protein was found to decrease by ~1 pH unit upon the introduction of one CF3 group, and the decrease in the pKa value with decreasing the PFQ value was confirmed by density functional theory calculations on some model compounds. The O2 affinity of Mb was found to correlate well with the pKa value in such a manner that the P50 value, which is the partial pressure of O2 required to achieve 50% oxygenation, increases by a factor of 2.7 with a decrease of 1 pKa unit. Kinetic studies on the proteins revealed that the decrease in O2 affinity upon the introduction of an electron-withdrawing CF3 group is due to an increase in the O2 dissociation rate. Since the introduction of a CF3 group substitution is thought to prevent further Fe2+-O2 bond polarization and hence formation of a putative Fe3+-O2--like species of the oxy form of the protein [Maxwell, J. C.; Volpe, J. A.; Barlow, C. H.; Caughey, W. S. Biochem. Biophys. Res. Commun. 1974, 58, 166-171], the O2 dissociation is expected to be enhanced by the substitution of electron-withdrawing groups as heme side chains. We also found that, in sharp contrast to the case of the O2 binding to the protein, the CO association and dissociation rates are essentially independent of the ρFe value. As a result, the introduction of electron-withdrawing group(s) enhances the preferential binding of CO to the protein over that of O2. These findings not only resolve the long-standing issue of the mechanism underlying the subtle tuning of the intrinsic heme Fe reactivity, but also provide new insights into the structure-function relationship of the protein. [ABSTRACT FROM AUTHOR]