Wilks A, Black S M, Miller W L, Ortiz de Montellano P R
Department of Pharmaceutical Chemistry, School of Pharmacy, University of California, San Francisco 94143-0446, USA.
Biochemistry. 1995 Apr 4;34(13):4421-7. doi: 10.1021/bi00013a034.
A human heme oxygenase (hHO-1) gene without the sequence coding for the last 23 amino acids has been expressed in Escherichia coli behind the pho A promoter. The truncated enzyme is obtained in high yields as a soluble, catalytically-active protein, making it available for the first time for detailed mechanistic studies. The purified, truncated hHO-1/heme complex is spectroscopically indistinguishable from that of the rat enzyme and converts heme to biliverdin when reconstituted with rat liver cytochrome P450 reductase. A self-sufficient heme oxygenase system has been obtained by fusing the truncated hHO-1 gene to the gene for human cytochrome P450 reductase without the sequence coding for the 20 amino acid membrane binding domain. Expression of the fusion protein in pCWori+ yields a protein that only requires NADPH for catalytic turnover. The failure of exogenous cytochrome P450 reductase to stimulate turnover and the insensitivity of the catalytic rate toward changes in ionic strength establish that electrons are transferred intramolecularly between the reductase and heme oxygenase domains of the fusion protein. The Vmax for the fusion protein is 2.5 times higher than that for the reconstituted system. Therefore, either the covalent tether does not interfere with normal docking and electron transfer between the flavin and heme domains or alternative but equally efficient electron transfer pathways are available that do not require specific docking.
一种缺失编码最后23个氨基酸序列的人血红素加氧酶（hHO-1）基因已在大肠杆菌中于pho A启动子后表达。截短的酶以高产量获得，是一种可溶的、具有催化活性的蛋白质，首次可用于详细的机制研究。纯化的截短hHO-1/血红素复合物在光谱上与大鼠酶的复合物无法区分，并且在用大鼠肝脏细胞色素P450还原酶重构时可将血红素转化为胆绿素。通过将截短的hHO-1基因与缺失编码20个氨基酸膜结合结构域序列的人细胞色素P450还原酶基因融合，获得了一个自给自足的血红素加氧酶系统。融合蛋白在pCWori+中的表达产生一种仅需NADPH即可进行催化周转的蛋白质。外源性细胞色素P450还原酶未能刺激周转以及催化速率对离子强度变化不敏感，这表明电子在融合蛋白的还原酶和血红素加氧酶结构域之间进行分子内转移。融合蛋白的Vmax比重构系统的Vmax高2.5倍。因此，要么共价连接不干扰黄素和血红素结构域之间的正常对接和电子转移，要么存在不需要特定对接的替代但同样有效的电子转移途径。
