Crystallization and structural analysis of binary complex of P450 electron donor and its electron transfer mechanism

通过EPR和ENDOR光谱学探测Pdx-P450摄像头二进制复合体的分子结构：在细胞色素P450摄像头进行单氧反应期间，一个P450摄像头复合体和减少的催乳素（Pdx）形成为反应的一个明显中间体。我们在其二进制复合体中发现了一种与摄像头绑定铁氧基P450摄像头的EPR光谱，明显地改变了Oy-D22、CO和NO到P450摄像头的铁氧基中心，并且在P450摄像头的Pdx绑定站点的Arg-112和109上发现了与此现象相关的重要角色。X-？D11？D1H-ENDOR results of the Pdx-P450cam binary complex demonstrated that the interactions of protons of cystein ligands with the unpaired electrons on the ironatoms in the reduced Pdx were altered upon CO binding to the ferrous heme of P450cam。我们还发现了一种与细胞色素P450 scc的二进制复合体的EPR谱，其二进制复合体已将CO绑定到P450 scc的铁基中，目前存在于20 S-羟基胆固醇，基础模拟中。精确分析正在进行中。Pdx-P450 cam二进制复合体的结晶化：一个无基础的P450 cam已经从以前报道的那样有不同的水晶形式的结晶化。单个晶体显微光度计被指示在此晶体中的基底-校园从未绑定到P450摄像头。此无基底P450摄像头的X射线晶体学分析，其位置的侧链附近的亚酸酯附近的亚基绑定站点已被显著地改变为与先前报告的分子结构相比较。我们也对Pdx-P450摄像头的二进制复合体产生了很大的影响。这个二进制复合体的水晶化直到现在还没有成功。“水晶化的影响正在进行中。”

Probing molecular structure of Pdx-P450cam binary complex by EPR and ENDOR spectroscopy : During the monooxygenation reaction by cytochrome P450cam, a complex of P450cam and reduced putidaredoxin (Pdx) is formed as an obligatory intermediate of the reaction. We found that EPR spectrum of the reduced Pdx in its binary complex with the camphor-bound ferrous P450cam was altered significantly upon ligation of OィイD22ィエD2, CO and NO to the ferrous heme of P450cam and that Arg-112 and 109 at the putative Pdx-binding site of P450cam played important roles associated with this phenomenon. X-band ィイD11ィエD1H-ENDOR results of the Pdx-P450cam binary complex demonstrated that the interactions of protons of cystein ligands with the unpaired electrons on the iron atoms in the reduced Pdx were altered upon CO binding to the ferrous heme of P450cam. We also found that the EPR spectrum of the reduced adrenodoxin (Adx) in its binary complex with cytochrome P450scc was altered upon CO binding to the ferrous heme of P450scc in the presence of 20S-hydroxy cholesterol, substrate analogue. Precise analyses are in progress.Crystallization of Pdx-P450cam binary complex : A substrate-free P450cam was crystallized having different crystalline form from that had been reported previously. Single crystal microspectrophotometry indicated that substrate-camphor never bound to P450cam in this crystal. X-ray crystallographic analysis of this substrate-free P450cam indicated that the positions of the side chains of the amino acid residues near the substrate-binding site were significantly altered as compared with the previously reported molecular structure. We have also made every effort to crystallize the binary complex of Pdx-P450cam. The crystallization of this binary complex is not succeeded until now. The efforts of the crystallization are in progress.