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

摘要

Abstract: Functional regulation of myoglobin (Mb) is thought to be achieved through the heme environmentnfurnished by nearby amino acid residues, and subtle tuning of the intrinsic heme Fe reactivity. We havenperformed substitution of strongly electron-withdrawing perfluoromethyl (CF3) group(s) as heme side chain(s)nof Mb to obtain large alterations of the heme electronic structure in order to elucidate the relationshipnbetween the O2 affinity of Mb and the electronic properties of heme peripheral side chains. We have utilizednthe equilibrium constant (pKa) of the “acid alkaline transition” in metmyoglobin in order to quantitativelynassess the effects of the CF3 substitutions for the electron density of heme Fe atom ( Fe) of the protein.nThe pKa value of the protein was found to decrease by ∼1 pH unit upon the introduction of one CF3 group,nand the decrease in the pKa value with decreasing the Fe value was confirmed by density functional theoryncalculations on some model compounds. The O2 affinity of Mb was found to correlate well with the pKanvalue in such a manner that the P50 value, which is the partial pressure of O2 required to achieve 50%noxygenation, increases by a factor of 2.7 with a decrease of 1 pKa unit. Kinetic studies on the proteinsnrevealed that the decrease in O2 affinity upon the introduction of an electron-withdrawing CF3 group is duento an increase in the O2 dissociation rate. Since the introduction of a CF3 group substitution is thought tonprevent further Fe2 O2 bond polarization and hence formation of a putative Fe3 O2 -like species of thenoxy form of the protein [Maxwell, J. C.; Volpe, J. A.; Barlow, C. H.; Caughey, W. S. Biochem. Biophys.nRes. Commun. 1974, 58, 166 171], the O2 dissociation is expected to be enhanced by the substitution ofnelectron-withdrawing groups as heme side chains. We also found that, in sharp contrast to the case of thenO2 binding to the protein, the CO association and dissociation rates are essentially independent of the Fenvalue. As a result, the introduction of electron-withdrawing group(s) enhances the preferential binding ofnCO to the protein over that of O2. These findings not only resolve the long-standing issue of the mechanismnunderlying the subtle tuning of the intrinsic heme Fe reactivity, but also provide new insights into thenstructure function relationship of the protein.