Henry E R, Rousseau D L, Hopfield J J, Noble R W, Simon S R
Biochemistry. 1985 Oct 8;24(21):5907-18. doi: 10.1021/bi00342a033.
Resonance Raman, optical absorption, and circular dichroism spectroscopic techniques have been used to examine the effect of the addition of inositol hexaphosphate (IHP) to a series of carp and human methemoglobin derivatives. Markers of spin equilibrium in the high-frequency region (1450-1650 cm-1) of the resonance Raman spectrum yield high/low-spin ratios consistent with direct magnetic susceptibility measurements. Changes in the low-frequency region (100-600 cm-1) of the resonance Raman spectrum appear to correlate with the quaternary structure transition. Changes in the ultraviolet absorption spectra and the circular dichroism spectra also appear to be related to the quaternary structure change. By using the resonance Raman spin markers, we find that those derivatives of carp methemoglobin which are in spin equilibrium have a larger ratio of high-spin to low-spin populations than the corresponding derivatives of human methemoglobin. Upon the addition of IHP to the methemoglobins the spin equilibrium is shifted toward a larger high-spin population. This change in equilibrium is larger for the carp protein than for the human protein. We obtain an IHP-induced change in the free energy difference between the high-spin and low-spin states of 300 cal/mol for those human methemoglobins in which a quaternary structure change occurs and 600 cal/mol for carp methemoglobins. Our data are consistent with a quaternary structure change induced by IHP in all the carp methemoglobins studied (F-, H2O, SCN-, NO2-, N3-, and CN-) and in the F-, H2O, and SCN- derivatives of the human protein but not in the NO2-, N3-, and CN- derivatives. The Fe-CN stretching mode has been identified by isotopic substitution and found to be unchanged in frequency in carp CN- metHb when the quaternary structure is changed. On the basis of our results we conclude that the protein forces at the heme due to the addition of IHP do not significantly affect the position of the iron atom with respect to the heme plane. Rather, the changes in spin equilibrium may be caused by protein-induced changes in the orientation of the proximal histidine or tertiary structure changes in the heme pocket which affect the porphyrin macrocycle. Either of these changes, or a combination thereof, leads to changes in the iron d orbital energies and concomitant changes in the spin equilibrium.
共振拉曼光谱、光吸收光谱和圆二色光谱技术已被用于研究向一系列鲤鱼和人高铁血红蛋白衍生物中添加肌醇六磷酸（IHP）的效果。共振拉曼光谱高频区域（1450 - 1650 cm⁻¹）中的自旋平衡标记给出的高/低自旋比与直接磁化率测量结果一致。共振拉曼光谱低频区域（100 - 600 cm⁻¹）的变化似乎与四级结构转变相关。紫外吸收光谱和圆二色光谱的变化似乎也与四级结构变化有关。通过使用共振拉曼自旋标记，我们发现处于自旋平衡的鲤鱼高铁血红蛋白衍生物的高自旋与低自旋群体比例比相应的人高铁血红蛋白衍生物更大。向高铁血红蛋白中添加IHP后，自旋平衡向更高的高自旋群体方向移动。鲤鱼蛋白的这种平衡变化比人蛋白的更大。对于发生四级结构变化的那些人高铁血红蛋白，我们得到IHP诱导的高自旋态和低自旋态之间自由能差的变化为300 cal/mol，对于鲤鱼高铁血红蛋白为600 cal/mol。我们的数据与IHP在所有研究的鲤鱼高铁血红蛋白（F⁻、H₂O、SCN⁻、NO₂⁻、N₃⁻和CN⁻）以及人蛋白的F⁻、H₂O和SCN⁻衍生物中诱导四级结构变化一致，但在NO₂⁻、N₃⁻和CN⁻衍生物中不一致。通过同位素取代确定了Fe - CN伸缩模式，发现在鲤鱼CN⁻高铁血红蛋白四级结构改变时其频率不变。根据我们的结果，我们得出结论，添加IHP后血红素处的蛋白质作用力不会显著影响铁原子相对于血红素平面的位置。相反，自旋平衡的变化可能是由蛋白质诱导的近端组氨酸取向变化或血红素口袋中的三级结构变化引起的，这些变化会影响卟啉大环。这些变化中的任何一种或它们的组合都会导致铁d轨道能量的变化以及自旋平衡的相应变化。
