Mechanism of porphyrin ring cleavage in the heme oxygenase catalysis

血红素加氧酶(HO)通过三次连续的氧化作用将血红素降解为胆绿素。虽然第三步氧化，即马鞭铁血红素的开环，被认为是体内调节HO酶活性的速率决定步骤，但在HO催化中，这一步骤一直被理解得最少。本研究项目的具体目的是通过详细的反应分析、结晶学和光谱表征来描述Ho的第三次氧化反应。在经典的HO反应中，我们发现HO通过O_2或H_2O_2双途径降解马鞭铁。结合抑制和突变研究，我们提出了以Fe-OH型马鞭铁作为反应中间体的机理。这一建议得到了以下事实的有力支持：小分子烷基过氧化氢(MeOOH和EtOOH)的大环裂解将烷基结合到线性四氢吡咯的一端。首次在肌红蛋白(一种血红素酶的模型蛋白)中表征了反应性过氧化物种。在1.0A的同步辐射下，氧肌红蛋白晶体在100K下被辐射还原，成功地生成了过氧肌红蛋白晶体。用0.6A同步辐射采集衍射数据，不需要过氧物种的进一步反应。化学合成了新的血红素分解代谢物S胆绿素，并通过LC-MS和核磁共振分析对其结构进行了鉴定。在新的HO反应中，马鞭铁的裂解是通过O2不依赖的方式进行的，S胆绿素与胆绿素的产物比例很大程度上取决于O2的浓度。这些机制的改变预计会影响HO的生理功能，即HO的O2敏感特性。

Heme oxygenase (HO) degrades heme to biliverdin through three successive oxygenations. While the third oxygenation, ring opening of verdoheme, is considered as the rate-determining step to regulate HO enzyme activity in vivo, this step has been the least understood in the Ho catalysis. Specific aim of this research project is to delineate the third oxygenation by HO through detailed reaction analysis, crystallographic and spectroscopic characterization. We also have examined a new HO reaction that affords a novel heme catabolite under physiologically relevant condition.In the classical HO reaction, we have found that HO degrades verdoheme through a dual pathway using either O_2 or H_2O_2. Together with inhibitory and mutational studies, we have proposed a mechanism involving an Fe-OOH verdoheme as a reactive intermediate. This proposal is strongly supported by the fact that the macrocycle cleavage with small alkyl hydroperoxides (MeOOH and EtOOH) incorporated the alkyl group into one end of linear tetrapyrroles produced.The reactive peroxy species is crystallographically characterized for the first time in myoglobin, a model protein of heme enzymes. Crystals of the peroxy myoglobin have been successfully generated by radiolytic reduction of the oxy myoglobin crystals at 100 K by irradiation with 1.0 A synchrotron radiation. Diffraction data is collected by 0.6 A synchrotron radiation without further reaction of the peroxy species.The new heme catabolite, S-biliverdin, has been chemically synthesized and identified by LC-MS and NMR analysis. The verdoheme cleavage in the new HO reaction proceeds through an O2-independent manner and the product ratio of S-biliverdin over biliverdin largely depends on the O2 concentration. These mechanistic changes are expected to affect on the physiological functions, namely an O2 sensing property of HO.