Huang Liusheng, Colas Christophe, Ortiz de Montellano Paul R
Contribution from the Department of Pharmaceutical Chemistry, University of California, 600 16th Street, San Francisco, CA 94143-2280, USA.
J Am Chem Soc. 2004 Oct 13;126(40):12865-73. doi: 10.1021/ja046455w.
Hemoproteins are powerful oxidative catalysts. However, despite the diversity of functions known to be susceptible to oxidation by these catalysts, it is not known whether they can oxidize carboxylic acids to carboxylic radicals. We report here that incubation of horseradish peroxidase (HRP) at acidic pH with H(2)O(2) in acetate buffer results in rapid modification of the heme group and loss of catalytic activity. Mass spectrometry and NMR indicate that an acetoxy group is covalently bound to the delta-meso-carbon in the modified heme. A heme with a hydroxyl group on the 8-methyl is also formed as a minor product. These reactions do not occur if protein-free heme and H(2)O(2) are co-incubated in acetate buffer, if the HRP reaction is carried out at pH 7, in the absence of H(2)O(2), or if citrate rather than acetate buffer is used. A similar heme modification is observed in incubations with n-caproic and phenylacetic acids. A mechanism involving oxidation of the carboxyl group to a carboxylic radical followed by addition to the delta-meso-position is proposed. This demonstration of the oxidation of a carboxylic acid solidifies the proposal that a carboxylic radical mediates the normal covalent attachment of the heme to the protein in the mammalian peroxidases and CYP4 family of P450 enzymes. The hemoprotein-mediated oxidation of carboxylic acids, ubiquitous natural constituents, may play other roles in biology.
血红素蛋白是强大的氧化催化剂。然而，尽管已知有多种功能易受这些催化剂氧化的影响，但尚不清楚它们是否能将羧酸氧化为羧基自由基。我们在此报告，在酸性pH条件下，将辣根过氧化物酶（HRP）与醋酸盐缓冲液中的H₂O₂一起孵育，会导致血红素基团迅速修饰并失去催化活性。质谱和核磁共振表明，一个乙酰氧基共价结合在修饰后血红素的δ-中位碳上。在8-甲基上带有羟基的血红素也作为次要产物形成。如果在醋酸盐缓冲液中共同孵育无蛋白的血红素和H₂O₂、如果HRP反应在pH 7下进行、在没有H₂O₂的情况下或如果使用柠檬酸盐缓冲液而非醋酸盐缓冲液，则不会发生这些反应。在用正己酸和苯乙酸孵育时也观察到类似的血红素修饰。提出了一种机制，即羧基先氧化为羧基自由基，然后加成到δ-中位位置。羧酸氧化的这一证明巩固了这样的提议，即羧基自由基介导了哺乳动物过氧化物酶和细胞色素P450酶的CYP4家族中血红素与蛋白质的正常共价连接。血红素蛋白介导的羧酸氧化，羧酸是普遍存在的天然成分，可能在生物学中发挥其他作用。
