Crystal structure of the cytochrome P450cam mutant that exhibits the same spectral perturbations induced by putidaredoxin binding
Crystal structure of the cytochrome P450cam mutant that exhibits the same spectral perturbations induced by putidaredoxin binding
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DOI：
10.1074/jbc.m404217200
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发表时间：
2004-10-08
影响因子：
4.8
通讯作者：
Poulos, TL
中科院分区：
文献类型：
--
作者：
Nagano, S;Tosha, T;Poulos, TL
众所周知，细胞色素P450cam活性位点会因与其氧化还原伙伴putidaredoxin （Pdx）结合而受到干扰。Pdx结合也增强了樟脑的单氧反应（Nagano， S., Shimada， H., Tarumi， A., Hishiki， T., Kimata- Ariga， Y., Egawa， T., Suematsu， M., Park， S.- Y., Adachi， S., Shiro， Y., and Ishimura， Y. (2003) Biochemistry 42,14507 - 14514）。这些作用是Pdx所特有的，因为非生理性电子供体不能支持樟脑单氧合。随附的H-1核磁共振论文(Tosha， T., Yoshioka， S., Ishimori， K., and Morishima， I. (2004) J. Biol.。Chem. 279, 42836 - 42843)表明L358P突变体的活性位点残基Thr-252和Cys-357以及铁（Fe(II)） CO配合物中的底物的构象与野生型酶与Pdx配合物的构象相似。为了探索这些变化是如何从pdx结合位点传递到活性位点的，我们已经解决了野生型和L358P突变体的铁和铁- co配合物的晶体结构。这些结构的比较表明，L358P突变导致Arg-112向血红素移动，Arg-112是一种已知对putidaredoxin结合很重要的残基。这种变化可以优化Pdx结合位点，从而提高Pdx的亲和力。突变还将血红素推向底物和配体结合袋，从而将底物重新定位到有利于区域选择性羟基化的位置。从较低的平均温度系数可以看出，樟脑的位置更加牢固。在I螺旋中催化重要的质子穿梭系统中涉及的残基也被突变改变。这种构象改变和突变氧配合物与非生理电子供体的反应性增强表明，Pdx结合优化了樟脑单氧化的远端口袋。
The cytochrome P450cam active site is known to be perturbed by binding to its redox partner, putidaredoxin (Pdx). Pdx binding also enhances the camphor monooxygenation reaction (Nagano, S., Shimada, H., Tarumi, A., Hishiki, T., Kimata- Ariga, Y., Egawa, T., Suematsu, M., Park, S.- Y., Adachi, S., Shiro, Y., and Ishimura, Y. ( 2003) Biochemistry 42, 14507 - 14514). These effects are unique to Pdx because nonphysiological electron donors are unable to support camphor monooxygenation. The accompanying H-1 NMR paper ( Tosha, T., Yoshioka, S., Ishimori, K., and Morishima, I. ( 2004) J. Biol. Chem. 279, 42836 - 42843) shows that the conformation of active site residues, Thr-252 and Cys-357, and the substrate in the ferrous ( Fe(II)) CO complex of the L358P mutant mimics that of the wild-type enzyme complexed to Pdx. To explore how these changes are transmitted from the Pdx-binding site to the active site, we have solved the crystal structures of the ferrous and ferrous-CO complex of wild-type and the L358P mutant. Comparison of these structures shows that the L358P mutation results in the movement of Arg-112, a residue known to be important for putidaredoxin binding, toward the heme. This change could optimize the Pdx-binding site leading to a higher affinity for Pdx. The mutation also pushes the heme toward the substrate and ligand binding pocket, which relocates the substrate to a position favorable for regio-selective hydroxylation. The camphor is held more firmly in place as indicated by a lower average temperature factor. Residues involved in the catalytically important proton shuttle system in the I helix are also altered by the mutation. Such conformational alterations and the enhanced reactivity of the mutant oxy complex with nonphysiological electron donors suggest that Pdx binding optimizes the distal pocket for monooxygenation of camphor.