title: Effect of heme modification on oxygen affinity of myoglobin and equilibrium of the acid-alkaline transition in metmyoglobin.
abstract: 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 perfluoromethyl (CF(3)) 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 O(2) affinity of Mb and the electronic properties of heme peripheral side chains. We have utilized the equilibrium constant (pK(a)) of the "acid-alkaline transition" in metmyoglobin in order to quantitatively assess the effects of the CF(3) substitutions for the electron density of heme Fe atom (rho(Fe)) of the protein. The pK(a) value of the protein was found to decrease by approximately 1 pH unit upon the introduction of one CF(3) group, and the decrease in the pK(a) value with decreasing the rho(Fe) value was confirmed by density functional theory calculations on some model compounds. The O(2) affinity of Mb was found to correlate well with the pK(a) value in such a manner that the P(50) value, which is the partial pressure of O(2) required to achieve 50% oxygenation, increases by a factor of 2.7 with a decrease of 1 pK(a) unit. Kinetic studies on the proteins revealed that the decrease in O(2) affinity upon the introduction of an electron-withdrawing CF(3) group is due to an increase in the O(2) dissociation rate. Since the introduction of a CF(3) group substitution is thought to prevent further Fe(2+)-O(2) bond polarization and hence formation of a putative Fe(3+)-O(2)(-)-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 O(2) 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 O(2) binding to the protein, the CO association and dissociation rates are essentially independent of the rho(Fe) value. As a result, the introduction of electron-withdrawing group(s) enhances the preferential binding of CO to the protein over that of O(2). 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.
authors.affiliation: Department of Chemistry, University of Tsukuba, Tsukuba 305-8571, Japan.
authors.forename: Tomokazu
authors.initials: T
authors.lastname: Shibata
authors.forename: Satoshi
authors.initials: S
authors.lastname: Nagao
authors.forename: Masashi
authors.initials: M
authors.lastname: Fukaya
authors.forename: Hulin
authors.initials: H
authors.lastname: Tai
authors.forename: Shigenori
authors.initials: S
authors.lastname: Nagatomo
authors.forename: Kenji
authors.initials: K
authors.lastname: Morihashi
authors.forename: Takashi
authors.initials: T
authors.lastname: Matsuo
authors.forename: Shun
authors.initials: S
authors.lastname: Hirota
authors.forename: Akihiro
authors.initials: A
authors.lastname: Suzuki
authors.forename: Kiyohiro
authors.initials: K
authors.lastname: Imai
authors.forename: Yasuhiko
authors.initials: Y
authors.lastname: Yamamoto
mesh_terms: D000143:Acids / Q000737:chemistry; D000468:Alkalies / Q000737:chemistry; D002248:Carbon Monoxide / Q000737:chemistry; D004583:Electrons; D006418:Heme / Q000737:chemistry* / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D008786:Metmyoglobin / Q000737:chemistry* / Q000378:metabolism; D009211:Myoglobin / Q000737:chemistry* / Q000378:metabolism; D019906:Nuclear Magnetic Resonance, Biomolecular; D010100:Oxygen / Q000378:metabolism*
chemical_list: D000143:Acids; D000468:Alkalies; D009211:Myoglobin; D008786:Metmyoglobin; D006418:Heme; D002248:Carbon Monoxide; D010100:Oxygen