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

详细信息    查看全文

• 作者：Tomokazu Shibata ; Satoshi Nagao ; Masashi Fukaya ; Hulin Tai ; Shigenori Nagatomo ; Kenji Morihashi ; Takashi Matsuo ; Shun Hirota ; Akihiro Suzuki ; Kiyohiro Imai ; Yasuhiko Yamamoto
• 刊名：Journal of the American Chemical Society
• 出版年：2010
• 出版时间：May 5, 2010
• 年：2010
• 卷：132
• 期：17
• 页码：6091-6098
• 全文大小：346K
• 年卷期：v.132,no.17(May 5, 2010)
• ISSN：1520-5126

文摘

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 (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 (pK_a) 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 (ρ_Fe) of the protein. The pK_a value of the protein was found to decrease by 1 pH unit upon the introduction of one CF₃ group, and the decrease in the pK_a value with decreasing the ρ_Fe value was confirmed by density functional theory calculations on some model compounds. The O₂ affinity of Mb was found to correlate well with the pK_a 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 pK_a 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 ρ_Fe 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.