The functionality of active centers in proteins is governed by the secondary and higher structure of proteins which often lead to structures in the active center that are different from the structures found in protein-free models of the active center. To elucidate this structure–function relationship, it is therefore necessary to investigate both the protein structure and the local structure of the active center. In this work, we investigate the application of hetero (resonance) Raman two-dimensional correlation spectroscopy (2D-COS) to this problem. By employing a combination of near-infrared-Fourier transform-Raman- and vis-resonance Raman spectroscopy, we could show that this combination of techniques is able to directly probe the structure–function relationship of proteins. We were able to correlate the transition of the heme center in cytochrome c from low to high spin with changes in the secondary structure with the above mentioned two spectroscopic in situ techniques and without sample preparation. Thereby, we were able to reveal that the combination of a spectroscopic method to selectively observe the active center with a technique that monitors the whole system offers a promising toolkit to investigate the structure–function relationship of proteins with photoactive centers in general.

蛋白质中活性中心的功能由蛋白质的二级和高级结构控制，这通常导致活性中心中的结构与活性中心无蛋白质模型中发现的结构不同。为了阐明这种结构-功能关系，有必要研究蛋白质结构和活性中心的局部结构。在这项工作中，我们研究了异质（共振）拉曼二维相关光谱 (2D-COS) 在这个问题上的应用。通过结合使用近红外傅立叶变换拉曼光谱和可见共振拉曼光谱，我们可以证明这种技术组合能够直接探测蛋白质的结构-功能关系。我们能够使用上述两种光谱原位技术将细胞色素 c 中血红素中心从低自旋到高自旋的转变与二级结构的变化相关联，无需样品制备。因此，我们能够揭示，选择性观察活性中心的光谱方法与监测整个系统的技术相结合，提供了一个很有前途的工具包，用于研究具有光活性中心的蛋白质的结构 - 功能关系。