Eltis L D, Karlson U, Timmis K N
Department of Microbiology, Gesellschaft für Biotechnologische Forschung, National Research Centre for Biotechnology, Braunschweig, Federal Republic of Germany.
Eur J Biochem. 1993 Apr 1;213(1):211-6. doi: 10.1111/j.1432-1033.1993.tb17750.x.
A soluble cytochrome P450 whose synthesis is induced by and that binds 2-ethoxyphenol was purified to apparent homogeneity from Rhodococcus rhodochrous strain 116. The enzyme had a subunit molecular mass of 44.5 kDa as determined by SDS/PAGE and a pI of 5.2. The electronic absorption spectrum indicates that the native cytochrome in the absence of substrate is predominantly in the low-spin state (13% high-spin state in 50 mM Mops, pH 7.0 25 degrees C). 2-Methoxyphenol binds to the cytochrome with a macroscopic dissociation constant of 0.53 +/- 0.03 microM (50 mM Mops, pH 7.0, 25 degrees C) and induces a 99.7% transition of the heme iron to the pentacoordinate high-spin form. Using a reconstituted in-vitro activity assay, it was demonstrated that P450RR1 catalyzed the O-dealkylation of 2-ethoxyphenol and 2-methoxyphenol to produce catechol. The cytochrome binds other ortho-substituted phenols, including 2-ethoxyphenol, 2-methylphenol (o-cresol) and 2-chlorophenol. The affinity of P450RR1 for these compounds is lower than that of 2-methoxyphenol and they are less effective than 2-methoxyphenol at inducing a transition in the heme iron to the high-spin state. Para-substituted and meta-substituted ether phenols did not induce a spin transition.
从红平红球菌116菌株中纯化出一种可溶性细胞色素P450，其合成由2-乙氧基苯酚诱导且能与2-乙氧基苯酚结合，纯化后的酶达到了表观均一性。通过SDS/PAGE测定，该酶的亚基分子量为44.5 kDa，pI为5.2。电子吸收光谱表明，在没有底物的情况下，天然细胞色素主要处于低自旋状态（在50 mM Mops，pH 7.0，25℃时，13%为高自旋状态）。2-甲氧基苯酚与细胞色素结合，其宏观解离常数为0.53±0.03 μM（50 mM Mops，pH 7.0，25℃），并诱导99.7%的血红素铁转变为五配位高自旋形式。使用重组体外活性测定法证明，P450RR1催化2-乙氧基苯酚和2-甲氧基苯酚的O-脱烷基反应生成邻苯二酚。该细胞色素还能结合其他邻位取代的酚类，包括2-乙氧基苯酚、2-甲基苯酚（邻甲酚）和2-氯苯酚。P450RR1对这些化合物的亲和力低于2-甲氧基苯酚，并且它们在诱导血红素铁向高自旋状态转变方面不如2-甲氧基苯酚有效。对位取代和间位取代的醚酚不会诱导自旋转变。
