Peroxygenase P450 as a Novel Bio-material

本课题的研究目的是了解枯草芽孢杆菌细胞色素P450BSβ的功能与结构之间的关系，该蛋白能以过氧化氢H_2O_2为氧供体催化脂肪酸的α-和β-位的羟基化。我们成功地制备了单晶，并在底物(棕榈酸)存在下测定了铁(Fe~(2+)~(2+))酶的结构(2·1Å分辨率)。在此结构的基础上，我们制备了一些突变体，并对它们的酶活性和光谱性质进行了测定，以揭示突变残基在酶功能(底物识别、催化反应、反应位点等方面的特异性)中的作用。提出了P450BS-β催化该反应的分子机理。在该机制中，Arg242与底物脂肪酸的羧酸盐结合，在过氧化氢O-O键断裂中起到普通酸碱催化剂的作用。测定了底物结合酶的CO络合物。结果发现，Phe79发生了移动，而其他的则没有与亚铁结合在一起。为了获得无底物形式的该酶晶体，我们研究了用凝胶过滤或用H_2O_2形成产物来去除结合底物的纯化方法，但我们还没有得到无底物形式的酶晶体。在对短命反应中间体(可能是Fe^&lt；5+&gt；=O)进行结构测定的过程中，我们试图使酶的氧络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。我们发现了氧络合物的部分形成。最后，我们详细地讨论了利用过氧化氢的含血红素酶的反应机理。

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.