Heme oxidation in a chimeric protein of the α-selective Neisseriae meningitidis heme oxygenase with the distal helix of the δ-selective
Heme oxidation in a chimeric protein of the α-selective Neisseriae meningitidis heme oxygenase with the distal helix of the δ-selective
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DOI：
10.1021/bi050810t
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发表时间：
2005-10-25
期刊：
影响因子：
2.9
通讯作者：
Wilks, A
中科院分区：
文献类型：
--
作者：
Deshmukh, R;Zeng, YH;Wilks, A
脑膜炎奈瑟菌(Nm-HO)和铜绿假单胞菌(pa-HO)的血红素加氧酶具有显著的序列同源性(37%)。在nm-140中，胆绿素IXα是反应的唯一产物，而pa-Ho主要产生胆绿素IX增量。我们先前已经通过核磁共振显示，由于血红素丙酸酯与蛋白质支架[Caignan，G.A.，Deshmukh，R.，Wilks，A.，Zeng，Y.，Huang，H.W.，Moenne-Loccoz，P.，Bunce，R.A.，Eastman，M.A.和Rivera，M.(2002)J.Am]的明显相互作用，PA-HO中的血红素的平面构象与nm-HO的平面构象显著不同。化学。SoC。14879-14892]。在这里提出的报告中，我们将这些研究扩展到通过制备nm-HO(nm-HOCH)嵌合体来研究远端螺旋的作用，其中nm-HO的远端螺旋残基107-142被Delta区域选择性的pa-HO(112-147)的相应残基取代。电子吸收光谱、共振拉曼光谱和FTIR光谱研究证实，与野生型蛋白质相比，嵌合体中His近端配体的取向和氢键性质没有明显改变。纳米-霍奇-血红素络合物的催化周转几乎只产生α-胆绿素和少量但可重复的三角洲-胆绿素。核磁共振谱研究表明，嵌合蛋白中区域选择性的改变可能源于血红素的两个交替平面构象之间的动态平衡(平面血红素紊乱)。在嵌合蛋白中，用Ala取代K16，用Lys取代Met31，以调节关键的多肽-血红素丙酸酯的接触，从而在很大程度上稳定了有利于Delta-Meso羟基化的平面构象。
Heme oxygenases from the bacterial pathogens Neisseriae meningitidis (nm-HO) and Pseudomonas aeruginosa (pa-HO) share significant sequence identity (37%). In nm-140, biliverdin IX alpha is the sole product of the reaction, whereas pa-HO yields predominantly biliverdin IX delta. We have previously shown by NMR that the in-plane conformation of the heme in pa-HO is significantly different from that of nm-HO as a result of distinct interactions of the heme propionates with the protein scaffold [Caignan, G. A., Deshmukh, R., Wilks, A., Zeng, Y., Huang, H. W., Moenne-Loccoz, P., Bunce, R. A., Eastman, M. A., and Rivera, M. (2002) J. Am. Chem. Soc. 124, 14879-14892]. In the report presented here, we have extended these studies to investigate the role of the distal helix by preparing a chimera of nm-HO (nm-HOch), in which distal helix residues 107-142 of nm-HO have been replaced with the corresponding residues of the delta-regioselective pa-HO (112-147). Electronic absorption spectra, resonance Raman and FTIR spectroscopic studies confirm that the orientation and hydrogen bonding properties of the proximal His ligand are not significantly altered in the chimera relative those of the wild-type proteins. The catalytic turnover of the nm-HOch-heme complex yields almost exclusively alpha-biliverdin and a small but reproducible amount of delta-biliverdin. NMR spectroscopic studies reveal that the altered regioselectivity in the chimeric protein likely stems from a dynamic equilibrium between two alternate in-plane conformations of the heme (in-plane heme disorder). Replacement of K16 with Ala and Met31 with Lys in the chimeric protein in an effort to tune key polypeptide-heme propionate contacts largely stabilizes the in-plane conformer conducive to delta-meso hydroxylation.