Department of Biological Chemistry, Medical School, University of Michigan, Ann Arbor, MI 48109-5606, USA.
Arch Biochem Biophys. 2010 Jan 15;493(2):184-91. doi: 10.1016/j.abb.2009.10.014. Epub 2009 Oct 30.
Our previous rapid-scanning stopped-flow studies of the reaction of substrate-free cytochrome P450cam with peracids [T. Spolitak, J.H. Dawson, D.P. Ballou, J. Biol. Chem. 280 (2005) 20300-20309; J. Inorg. Biochem. 100 (2006) 2034-2044; J. Biol. Inorg. Chem. 13 (2008) 599-611] spectrally characterized compound I (ferryl iron plus a porphyrin pi-cation radical (Fe(IV)O/Por(.+))), Cpd ES, and Cpd II (Fe(IV)O/Tyr() or Fe(IV)O). We now report that reactions of CYP153A6 with peracids yield all these intermediates, with kinetic profiles allowing better resolution of all forms at pH 8.0 compared to similar reactions with WT P450cam. Properties of the reactions of these higher oxidation state intermediates were determined in double-mixing experiments in which intermediates are pre-formed and ascorbate is then added. Reactions of heptane-bound CYP153A6 (pH 7.4) with mCPBA resulted in conversion of P450 to the low-spin ferric form, presumably as heptanol was formed, suggesting that CYP 153A6 is a potential biocatalyst that can use peracids with no added NAD(P)H or reducing systems for bioremediation and other industrial applications.
我们之前的快速扫描停流研究表明,在没有底物的细胞色素 P450cam 与过酸反应中[T. Spolitak、J.H. Dawson、D.P. Ballou,J. Biol. Chem. 280 (2005) 20300-20309;J. Inorg. Biochem. 100 (2006) 2034-2044;J. Biol. Inorg. Chem. 13 (2008) 599-611],光谱学上鉴定了化合物 I(铁酰基加上卟啉 π-阳离子自由基(Fe(IV)O/Por(.+)))、Cpds ES 和 Cpd II(Fe(IV)O/Tyr()或 Fe(IV)O)。现在我们报告说,CYP153A6 与过酸的反应产生了所有这些中间体,动力学谱图允许在 pH 8.0 下更好地分辨所有形式,与 WT P450cam 的类似反应相比。在双混合实验中确定了这些更高氧化态中间体的反应性质,其中中间体预先形成,然后加入抗坏血酸。在 pH 7.4 的庚烷结合的 CYP153A6 与 mCPBA 的反应中,P450 转化为低自旋三价铁形式,可能是因为形成了庚醇,这表明 CYP 153A6 是一种潜在的生物催化剂,可以在没有添加 NAD(P)H 或还原系统的情况下使用过酸进行生物修复和其他工业应用。