Peroxygenase P450 as a Novel Bio-material

本课题的研究目的是了解枯草芽孢杆菌（Bacillus subtilis）细胞色素P450Bsβ在过氧化氢H_2O_2供氧条件下催化脂肪酸α-和β-羟基化的功能和结构关系。我们成功地制作了单晶，然后在底物（棕榈酸）存在下确定了铁（Fe^<3+>）酶的结构（2.1Å分辨率）。在此基础上，我们制备了一些突变体，并测量了它们的酶活性和光谱性质，以揭示突变残基在酶功能中的作用（在底物识别、催化反应、反应位点等方面的特异性）。提出了p450bs - β催化反应的分子机理。在其机制中，Arg242与底物脂肪酸的羧酸结合，在过氧化物O-O键的裂解中起一般酸碱催化剂的作用。测定了底物结合酶的CO配合物。发现Phe79被移动了，但其他的没有被CO与亚铁结合。为了获得无底物形式的该酶晶体，我们研究了通过凝胶过滤或与H_2O_2形成产物去除结合底物的纯化程序。但是我们还没有得到无底物酶的晶体。在进行短寿命反应中间体（可能是Fe^<5+>=O态）的结构测定时，我们尝试将酶的氧配合物稳定在溶液状态，然后用x射线还原技术注入两个电子。我们发现了氧配合物的部分形成。最后讨论了含血红素酶利用过氧化氢的反应机理。

Our research aim in this project was to understand the relationship of function and structure of cytochrome P450Bsβ from Bacillus subtilis, which can catalyze the hydroxylation of α- and β-positions of fatty acids using hydrogen peroxide H_2O_2 as an oxygen donor. We succeeded in making the single crystal and afterward in determining the structure (2.1Å resolution) of the ferric (Fe^<3+>) enzyme in the presence of a substrate (palimitic acid). On the basis of the structure, we prepared some mutants, and measured their enzymatic activities and spectral properties, to reveal roles of the mutated residues in the enzymatic functions (specificities in the substrate recognition, the catalytic reactions, the reaction sites, etc.). Then we proposed the molecular mechanism of the reaction catalyzed by P450Bsβ. In the mechanism, Arg242 binds with the carboxylate of the substrate fatty acid, which acts as a general acid-base catalyst in the peroxide O-O bond cleavage. The CO complex of the substrate-bound enzyme was determined. It was found that Phe79 was moved, but others were not upon the CO binding to the ferrous iron. To obtain a crystal of this enzyme in the substrate-free form, we examined the purification procedures for removal of the bound substrate by gel-filteration or by the product formation with H_2O_2. But we have not yet obtained the crystal of the substrate-free enzyme. In pursuing the structural determination of the short-lived reaction intermediate (possibly the Fe^<5+>=O state), we tried to stabilize the oxy complex of the enzyme in solution state, and then to inject two electrons with the X-ray reduction technique. We found the partial formation of the oxy complex. Finally, we discussed details of the reaction mechanism of heme-containing enzymes which utilize hydrogen peroxide.