Time-Resolved Vibrational Spectroscopic Investigation of Ultrafast Protein Dynamics Coupled with Photoreaction

在许多生物系统中，局部的微小结构变化在空间上延伸到介观维度以实现生理功能，而蛋白质的高阶结构变化对这一过程是必不可少的。肌红蛋白(Mb)是研究蛋白质这一特性的最好分子之一，因为CO从一氧化碳Mb(MbCO)的光解离是一种局域反应，其量子产率几乎为1。X-射线研究表明，CO脱除后，铁原子移出卟啉平面约0.3A，核心尺寸膨胀0.05A。为了探索这一过程中涉及的蛋白质动力学，我们研究了光解MbCO的皮秒时间分辨共振拉曼光谱。在1300-1650 cm~(-1)&gt~(-1)区域对CO光解后的脱氧Mb光谱的仔细检查表明，在仪器响应时间(~2ps)内，卟啉环的核心尺寸的扩展完成。相反，Fe-His伸缩模式(VFe-His)的强度和频率在皮秒内发生变化，这表明Fe从卟啉平面的位移具有明显的时间演化。没有蛋白质基质的血红素基团的模型化合物也有同样的行为。因此，这反映了血红素本身的一种内在属性。另一方面，VFe-His模的频率随时间常数的变化约为100ps。对于没有蛋白质基质的模型化合物，这种频率变化不明显。因此，这一定是由蛋白质的三级结构变化引起的。V4和V7谱带的反斯托克斯拉曼强度随时间的变化表明，在光解过程中立即产生了振动激发的血红素，随后振动激发态的能级数衰减，时间常数分别为1.1±0.6ps和1.9±0.6ps。

In many biological systems a localized small structural change extends spatially to mesoscopic dimensions to achieve a physiological function, and higher-order structural changes of proteins are essential to this process. Myoglobin (Mb) is one of the best molecules for studying such features of proteins, because photodissociation of CO from carbonmonoxyMb (MbCO), a localized reaction, takes place within 50 fs like a step-function with a quantum yield of nearly unity. It is know from x-ray studies that the iron atom moves out of the porphyrin plane by 〜0.3 A and the core-size expands by 0.05A upon deligation of CO. To explore the protein dynamics involved in this process, we have investigated picosecond time-resolved resonance Raman spectra of photodissociated MbCO.Close inspection of the spectra of deoxyMb following CO photolysis in the 1300-1650 cm^<-1> region revealed that the core-size expansion of porphyrin ring is completed within the instrumental response time (〜2 ps). In contrast, changes in the intensity and frequency of the Fe-His stretching mode (VFe-His) occurred in picoseconds, suggesting appreciable time evolution for the Fe displacement from the porphyrin plane. The same behaviors were observed for the model compound of the heme group without protein matrix. Therefore, this reflects an intrinsic property of heme itself. On the other hand, the frequency of the VFe-His mode changed with a time constant of 〜100 ps. This frequency change was not seen for the model compound without the protein matrix. Therefore, this must be caused by tertiary structural changes of the protein. Temporal changes of the anti-Stokes Raman intensity of the V_4 and V_7 bands demonstrated immediate generation of the vibrationally excited heme upon photolysis and subsequent decay of the vibrationally excited population with the time constants of 1.1±0.6 and 1.9±0.6 ps, respectively.