Mechanism of heme degradation by heme oxygenase and the interaction of heme

血红素加氧酶通过三个顺序的单加氧酶反应催化氯化血红素区域特异性氧化为胆绿定IXα，同时释放CO和铁。(1)我们用EPR研究了血红素转化为α-meso-羟基血红素的反应。ENDOR和光学吸收光谱。这些研究结果表明，过氧化氢铁HO确实是反应活性物种，通过过氧化氢部分的末端OH攻击血红素α-碳直接形成α-meso-羟基血红素。D140 A突变体不能将血红素转化为α-meso-hydroxyheme，证实了我们先前的发现，即HO远端口袋内的氢键网络被破坏。(2)我们研究了从meso-羟基氯化血红素到verdoheme和versoheme到胆绿素的两个反应步骤中的每一个的立体选择性，通过使用大鼠HO-1的截短形式和化学合成的meso-羟基氯化血红素和verdoheme的四个异构体。HO-1将内消旋羟基氯化血红素的所有四种异构体转化为相应的 关于我们 绿血红素的异构体。相反，只有绿血红素IXα转化为相应的胆绿素IXα。我们的结论是，第三步，而不是第二步，是立体选择性的α-异构体底物。(3)利用人工合成的HO-1基因构建了蓝藻集胞藻6803 HO基因的高效表达系统。可溶性蛋白在高水平表达，并高度纯化，为第一次。光谱特征以及分解代谢活性强烈表明，尽管一级结构非常保守，但集胞藻HO-1的血红素口袋结构不同于大鼠HO。(4)白喉棒状杆菌血红素O（HemO）的铁和亚铁血红素络合物的晶体结构已分别精确到1.4和1.5埃分辨率。血红素口袋结构负责通过防止过早水解O-O键断裂来稳定铁氢过氧活性中间体。少

Heme oxygenase catalyzes the regiospecific oxidation of hemin to biliverding IXα with concomitant liberation of CO and iron by three sequential mono-oxygenase reaction. (1) We studied the conversion of heme to α-meso-hydroxyheme by EPR. ENDOR, and optical absorption spectroscopy. The results obtained by these studies demonstrate that hydroperoxo ferric-HO is indeed the reactive species, directly forming the α-meso-hydroxyheme by attack of the distal OH of hydroperoxo moiety at the heme α-carbon. D140A mutant failed to convert heme to α-meso-hydroxyheme, confirming our previous finding that the H-bonding network within the distal pocket of HO is disrupted. (2) We investigated the stereoselectivity of each of the two reaction steps from meso-hydroxyhemin to verdoheme and versoheme to biliverdin, by using a truncated form of rat HO-1 and the chemically synthesized four isomers of meso-hydroxythemin and verdoheme. HO-1 converted all four isomers of meso-hydroxyhemin to the corresponding is … More omers of verdoheme. In contrast, only verdoheme IXα was converted to the corresponding biliverdin IXα. We conclude that the third step, but not the second, is stereoselective for the α-isomer substrate. (3) An efficient bacterial expression system of cyanobacterium Synechocystis sp. PCC 6803 HO gene, ho-1, has been constructed, using a synthetic gene. A soluble protein was expressed at high levels and was highly purified, for the first time. The spectroscopic characters as well as the catabolic activities strongly suggest that, in spite of very high conservation of the primary structure, the heme pocket structure of Synechocystis HO-1 is different from that of rat HO. (4) Crystal structure of the ferric and ferrous heme complexes of HemO, a 24-kDA HO of Corynebacterium diphtheriae, have been refined to 1.4 and 1.5 A resolustion, respectively. The heme pocket architecture is responsible for stabilization of the ferric hydroperoxo-active intermediated by preventing premature hydrolytic O-O bond cleavage. Less