光谱法研究细胞色素C与proliNONOate之间的相互作用

细胞色素C(Cytochrome，Cyt c)作为细胞内线粒体的电子传递体，与NO之间的相互作用结果对于线粒体凋亡的检测有着重要的生物学意义。采用紫外-可见(UV-Vis)吸收光谱、电子顺磁技术(EPR)、时间过程光谱、圆二色(CD)光谱和同步荧光光谱等方法对处于不同价态的Cyt c与NO替代物，即proliNO/NO(proliNONOate)的相互作用过程，以及Cyt c在结合NO过程中蛋白空间结构的变化进行了详细研究。实验结果发现： 在模拟生理条件下，不同价态的Cyt c都能够直接与NO替代物(proliNO/NO)所产生的NO发生配位反应。推断出Cyt c与NO之间相互作用发生的可能机制： NO与Cyt c结合过程，是因为与Cyt c中Fe配位的轴向配体以及卟啉周围的取代基变化而发生结合的。具体反应过程为： 溶液中的NO诱导Cyt c中的Heme上Met80远离原来位置，Fe-S键断裂，进而空出的Fe与NO结合生成Fe-N键，从而生成新的Cyt c-NO配合物。研究表明： Cyt c-NO二元复合物不稳定，会发生光解离反应，通过线性拟合得出： 其解离过程属于准一级反应，解离速率为(0.071 1±0.039 6) s-1。同时，血红素Fe与NO间新键的形成，影响了色氨酸和酪氨酸微环境的变化；Cyt c二级结构受proliNO/NO浓度的影响，当proliNO/NO浓度低于8.6×10-4 mol·L-1时，Cyt c的α-螺旋特征峰强度变化很小，且位置不变；但proliNO/NO浓度高于8.6×10-4 mol·L-1时，即过量，Cyt c的二级结构变化很明显，说明过量的NO能导致Cyt c二级结构的破坏。NO与Cyt c配位反应机制的研究对于利用NO抑制线粒体内的氧气消耗，以及线粒体内NO的代谢具有重要的意义。

Spectral Study on the Interaction between Cytochrome c and ProliNONOate

It played an important role in the detection of mitochondrial apoptosis that the interaction of Cytochrome(Cyt c) with NO, thus the research is still a hotspot issue for the chemists and biologists. In this paper, Ultraviolet (UV-Vis) absorbance spectra, UV-Vis time course spectra, circular dichroism (CD), synchronous fluorescence spectra and electron paramagnetic resonance (EPR) technology were used to research the coordination reaction mechanism of different valence states Cyt c with proliNO/NO and its spatial conformation changes. The results showed that Cyt c could directly react with NO without other reagents. And its secondary structure would change during the experiment. It has been established that the electronic configurations of iron ions in porphyrin complexes are controlled in the nature by a number of axial ligands, peripheral substituents attached to the porphyrin macrocycle, deformation of the porphyrin ring and solvent effects. During the experiment, proliNO/NO was added to the sample of Cyt c, and NO gas would generate, then entered into the solution, finally the distal methionine heme ligand was displaced, Cyt c could bind NO, that is, the Fe-S broke down, and the Fe-N formed. At the same time, generating a new cytochrome c-NO (Cyt c-NO) complex. Cyt c-NO binary complexes were instability and would dissociate, and dissociation process of Cyt c-NO binary complexes was belonging to a first-order reaction with the dissociation rate of (0.071 1±0.039 6) s-1. The secondary structure of Cyt c was affected by proliNO/NO concentration. When the concentrate of proliNO/NO was below to 8.6×10-4 mol·L-1, the peak changes of 222 nm and 208 nm was very weak, and the α-helix increased from 33.1% to 44.1%. And it continued to increase, the secondary structure of Cyt c took place a great change. The tiny changes illustrated that a new compound generated, but excessive proliNO/NO can break the structure of Cyt c. Taken together, we have demonstrated that an understanding of the interaction mechanism of NO with cytc, and the reaction mechanism of NO with ferri- and ferro-cytochrome c have important implications for the inhibition of mitochondrial oxygen consumption by NO and the mitochondrial metabolism of NO.