Sono M, Smith P D, McCray J A, Asakura T
J Biol Chem. 1976 Mar 10;251(5):1418-26.
In order to study the effects of chemical modifications of the vinyl groups of heme on oxygen and carbon monoxide binding to myoglobin, apomyoglobins from horse heart were reconstituted with six different hemins with various side chains. Laser flash photolysis experiments of these reconstituted myoglobins showed that the combination rate constants for oxygen (k') and carbon monoxide (l') were closely related to the electron-attractive properties of the side chains. The k' values obtained in 0.1 M potassium phosphate buffer, pH 7.0, at 20 degrees were 0.83 (meso-), 2.4 (deutero-), 1.1 (reconstituted proto-), 1.2 (native proto-), 1.5 (2-formyl-4-vinyl-), 1.9 (2-vinyl-4-formyl-), and 2.7 X 10(7) M-1 S-1 (2,4-diformylmyoglobins), and the corresponding l' values were 2.8, 18, 4.8, 5.1, 7.1, 15, and 35 X 10(5) M-1 S-1, respectively. These rate constants tend to increase as the electron-withdrawing power of the side chains increases, indicating that reduced electron density of the iron atom of heme in myoglobin favors the combination reaction for both oxygen and carbon monoxide. Equilibrium constants (L) between carbon monoxide and various myoglobins were also determined by measuring the partition coefficients (M) between oxygen and carbon monoxide for the myoglobins, and were also found to be closely related to the electronic properties (pK3 of porphyrin) of the heme side chains. The equilibrium association constants for carbon monoxide thus obtained increased with a decrease in pK3 value of the porphyrin. This order was completely opposite to the case of the oxygen binding reaction. The dissociation rate constants for oxygen (k) and carbon monoxide (l) were calculated from the equilibrium and the combination rate constants. The dissociation rate constants showed a similar characteristic to the combination rate constants and increased with the increase in electron attractivity of heme side chains. The concomitant increase in both the combination and dissociation rate constants with increase in electronegativity of the iron atom suggests that these reactions have different rate determining steps, although such a reaction process is contradictory to the generally accepted concept that in a reversible reaction, both on and off reactions proceed through the same transition state. In the on reaction sigma bond formation appears to be dominant, while in the off reaction eta bond break-up is more important.
为了研究血红素乙烯基化学修饰对氧和一氧化碳与肌红蛋白结合的影响，用六种具有不同侧链的不同血红素对马心脱辅基肌红蛋白进行了重组。对这些重组肌红蛋白进行的激光闪光光解实验表明，氧（k'）和一氧化碳（l'）的结合速率常数与侧链的吸电子性质密切相关。在0.1 M磷酸钾缓冲液（pH 7.0，20℃）中获得的k'值分别为0.83（中位-）、2.4（次位-）、1.1（重组原卟啉-）、1.2（天然原卟啉-）、1.5（2-甲酰基-4-乙烯基-）、1.9（2-乙烯基-4-甲酰基-）和2.7×10⁷ M⁻¹ s⁻¹（2,4-二甲酰基肌红蛋白），相应的l'值分别为2.8、18、4.8、5.1、7.1、15和35×10⁵ M⁻¹ s⁻¹。这些速率常数倾向于随着侧链吸电子能力的增加而增加，这表明肌红蛋白中血红素铁原子的电子密度降低有利于氧和一氧化碳的结合反应。还通过测量肌红蛋白中氧和一氧化碳之间的分配系数（M）来确定一氧化碳与各种肌红蛋白之间的平衡常数（L），并且发现其也与血红素侧链的电子性质（卟啉的pK3）密切相关。由此获得的一氧化碳平衡缔合常数随着卟啉pK3值的降低而增加。这个顺序与氧结合反应的情况完全相反。根据平衡常数和结合速率常数计算出氧（k）和一氧化碳（l）的解离速率常数。解离速率常数表现出与结合速率常数相似的特征，并且随着血红素侧链吸电子性的增加而增加。随着铁原子电负性的增加，结合和解离速率常数同时增加，这表明这些反应具有不同的速率决定步骤，尽管这样的反应过程与普遍接受的概念相矛盾，即在可逆反应中，结合和解离反应都通过相同的过渡态进行。在结合反应中，σ键形成似乎占主导，而在解离反应中，η键断裂更为重要。
