Srajer V, Champion P M
Department of Physics, Northeastern University, Boston, Massachusetts 02115.
Biochemistry. 1991 Jul 30;30(30):7390-402. doi: 10.1021/bi00244a005.
We present the results of an extensive investigation of the optical line shapes of deoxymyoglobin (Mb), the ligand-bound form (MbCO), and the low-temperature photoproduct (Mb*). The thermal properties and the pH dependence of the Soret band and the near infrared band III (approximately 760 nm) are analyzed, taking into account the underlying vibrational properties of the absorption bands. The strong temperature dependence associated with the Soret band of MbCO and band III of Mb indicates significant coupling to low-frequency modes that may not be directly observed in the resonance Raman spectra. On the basis of analogous line-shape studies in a variety of heme systems, we assign the low-frequency coupling in MbCO to torsional motions of the CO molecule. The low-frequency mode coupled to band III (approximately 70 cm-1) is found to lie quite close to the value for the heme-doming motion (approximately 50 cm-1) calculated by using the kinetically determined value of the force constant (17 N/m). Significant inhomogeneous broadening in the Soret region of Mb and Mb* is found to be due to a "nonkinetic" coordinate that we associate with the orientation of the proximal histidine. A "kinetic" coordinate, associated with the equilibrium displacement of the iron atom from the porphyrin plane (a) is found to contribute to the inhomogeneous broadening of both the Soret band and band III. The relaxation of the heme as the system evolves from from Mb* to Mb is followed optically as a function of temperature, and a sharp transition temperature is found at 185 K. The blue shifts of the Soret band and band III as Mb* evolves to Mb are found to be nearly identical (delta vABS approximately 140 cm-1) and attributed to changes in the mean value of a between Mb (a0) and Mb (a0 = 0.45 A). A simple quadratic model for the coordinate coupling that simultaneously accounts for the observed shift, delta vABS, the low-temperature kinetics and the kinetic hole burning predicts a*0 = 0.2 +/- 0.05 A and EA = 16 +/- 2 kJ/mol for the room temperature Arrhenius barrier height at the heme. A simple quantitative method for the analysis of kinetic hole-burning experiments is also developed and applied to recent studies involving quaternary and subunit-specific hemoglobin structures.
我们展示了对脱氧肌红蛋白（Mb）、配体结合形式（MbCO）和低温光产物（Mb*）的光谱线形进行广泛研究的结果。考虑到吸收带的潜在振动特性，分析了索雷特带和近红外带III（约760 nm）的热性质和pH依赖性。与MbCO的索雷特带和Mb的带III相关的强烈温度依赖性表明与低频模式存在显著耦合，而这种耦合在共振拉曼光谱中可能无法直接观察到。基于对各种血红素系统类似线形研究的基础上，我们将MbCO中的低频耦合归因于CO分子的扭转运动。发现与带III耦合的低频模式（约70 cm⁻¹）非常接近通过动力学确定的力常数（17 N/m）计算出的血红素穹顶运动的值（约50 cm⁻¹）。发现在Mb和Mb的索雷特区域存在显著的非均匀展宽，这是由于我们将其与近端组氨酸的取向相关联的一个“非动力学”坐标引起的。一个与铁原子从卟啉平面的平衡位移（a）相关的“动力学”坐标，被发现对索雷特带和带III的非均匀展宽都有贡献。随着系统从Mb演变为Mb，血红素的弛豫过程通过光学手段跟踪其随温度的变化，发现在185 K处有一个尖锐的转变温度。当Mb演变为Mb时，索雷特带和带III的蓝移被发现几乎相同（ΔvABS约140 cm⁻¹），并归因于Mb*（a0）和Mb（a0 = 0.45 Å）之间a的平均值的变化。一个用于坐标耦合的简单二次模型，它同时考虑了观察到的位移ΔvABS、低温动力学和动力学空穴烧蚀，预测在室温下血红素的阿仑尼乌斯势垒高度a*0 = 0.2 ± 0.05 Å和EA = 16 ± 2 kJ/mol。还开发了一种用于分析动力学空穴烧蚀实验的简单定量方法，并将其应用于最近涉及四级和亚基特异性血红蛋白结构的研究中。
