Wilks A, Ortiz de Montellano P R
Department of Pharmaceutical Chemistry, School of Pharmacy, University of California, San Francisco 94143-0446.
J Biol Chem. 1993 Oct 25;268(30):22357-62.
A rat heme oxygenase (HO-1) gene without the sequence coding for the last 23 amino acids has been constructed and expressed behind the pho A promoter in Escherichia coli. The enzyme is expressed at high levels as a soluble catalytically active protein that causes the bacterial cells to accumulate biliverdin. The purified truncated heme-heme oxygenase complex is spectroscopically indistinguishable from the complex with the native enzyme and converts heme to biliverdin when reconstituted with rat liver cytochrome P450 reductase. Reaction of the recombinant heme-heme oxygenase complex with H2O2 produces a species with the spectroscopic properties of verdoheme. Unidentified products are obtained when this intermediate is directly extracted from the protein, but biliverdin is obtained if the verdoheme-protein complex is exposed to cytochrome P450 reductase and NADPH before the extraction step. In contrast, reaction of the heme-heme oxygenase complex with meta-chloroperbenzoic acid (mCPBA), tert-butylhydroperoxide, or cumene hydroperoxide yields a ferryl (FeIV = O) complex. Reaction of the heme-heme oxygenase complex with mCPBA also produces an EPR-detectable protein radical. In accord with formation of a ferryl intermediate, recombinant heme oxygenase catalyzes the mCPBA- and alkylhydroperoxide-dependent peroxidation of 2-methoxyphenol (guaiacol). Guaiacol oxidation is not observed during turnover of the enzyme by cytochrome P450 reductase/NADPH or H2O2. Conversely, biliverdin is not formed with tert-butylhydroperoxide or mCPBA. H2O2 thus supports the first step of the normal catalytic oxidation of heme by heme oxygenase, but alkyl and acyl hydroperoxides do not. These results suggest that the alpha-meso-hydroxylation required for biliverdin formation is mediated by the distal of the two oxygens in the iron-dioxygen intermediate (Fe-O-O) engendered by reaction with either cytochrome P450 reductase/NADPH or H2O2.
已构建了一个缺少编码最后23个氨基酸序列的大鼠血红素加氧酶（HO-1）基因，并在大肠杆菌的pho A启动子后表达。该酶以高水平表达为一种可溶性的具有催化活性的蛋白质，导致细菌细胞积累胆绿素。纯化的截短型血红素-血红素加氧酶复合物在光谱上与天然酶的复合物无法区分，并且在用大鼠肝细胞色素P450还原酶重构时能将血红素转化为胆绿素。重组血红素-血红素加氧酶复合物与H2O2反应产生一种具有胆绿血红素光谱特性的物质。当这种中间体直接从蛋白质中提取时会得到未鉴定的产物，但如果在提取步骤之前将胆绿血红素-蛋白质复合物暴露于细胞色素P450还原酶和NADPH，则会得到胆绿素。相反，血红素-血红素加氧酶复合物与间氯过苯甲酸（mCPBA）、叔丁基过氧化氢或异丙苯过氧化氢反应会产生一个高铁（FeIV = O）复合物。血红素-血红素加氧酶复合物与mCPBA反应还会产生一个可通过电子顺磁共振检测到的蛋白质自由基。与高铁中间体的形成一致，重组血红素加氧酶催化mCPBA和烷基过氧化氢依赖的2-甲氧基苯酚（愈创木酚）的过氧化反应。在细胞色素P450还原酶/NADPH或H2O2使酶周转的过程中未观察到愈创木酚氧化。相反，叔丁基过氧化氢或mCPBA不会形成胆绿素。因此，H2O2支持血红素加氧酶对血红素正常催化氧化的第一步，但烷基和酰基过氧化氢则不支持。这些结果表明，胆绿素形成所需的α-中位羟基化是由与细胞色素P450还原酶/NADPH或H2O2反应产生的铁-双氧中间体（Fe-O-O）中两个氧原子的远端介导的。
