硫化氢激活EcDOS及其化学修饰血红素机制研究生物化学与分子生物学专业论文.docxVIP

汕头大 汕头大学医学院硕士学位论文 II II 和 R97I 这两个突变蛋白与 H2S 的反应中被检测到。中间产物过氧化氢也被证实在反应过 程中产生。（5）R97 位点的突变使 Ec DOS 血红素结合结构域的血红素结合部位之构象接 近于血红素加氧酶的相应活性区域。这一改变赋予 Ec DOS 血红素结合结构域新的功能。 关键词：氧感应蛋白；硫化氢；气体信号分子；血红素加氧酶 III III ABSTRACT Direct Oxygen Sensor from E. coli (Ec DOS), one of the essential regulators in the c-di-GMP related signaling pathways of E. coli, has been found a high c-di-GMP catalytic activity which enables its c-di-GMP metabolism activity. The previous studies confirmed that this enzyme can be activated by oxygen binding. And then, NO and CO were reported their activations of Ec DOS by binding to it. The novel gasotransmitter, hydrogen sulfide (H2S) was also newly reported its activation of Ec DOS catalytic via binding to the heme iron inside Ec DOS. While, different from NO and CO which bind to ferrous (Fe2+) heme, H2S only binds to ferric (Fe3+) heme. What’s more, H2S was discovered the significantly higher activation of Ec DOS enzyme activity under aerobic than anaerobic condition. All of these suggest the different mechanisms of reactions between NO/CO and H2S with heme inside Ec DOS, perhaps more complex in the case of H2S. The present thesis contains the detailed descriptions on how the mechanism of Ec DOS activation by H2S was discovered and what was revealed in this study by using UV-Vis Spectroscopy and Mass Spectrum. Site directed mutations was adopted to examine the effects of the two heme-protein interaction sites, M95 and R97, locates at the heme-protein interaction region in the isolated heme domain of Ec DOS. UV-Vis spectra differences between the wild type protein and the mutants would uncover the beneath mechanism. The results obtained in this study by UV-Vis spectroscopy confirmed both of the two sites are essential for the interaction, while R97 is even more important, because it is the residue interacts with heme bound ligand directly. Mutations at R97 totally deprive the reaction activity of Ec DOS with H2S, and destabilize the protein structure. Mutatio