Kuo C L, Raner G M, Vaz A D, Coon M J
Department of Biological Chemistry, Medical School, The University of Michigan, Ann Arbor 48109-0606, USA.
Biochemistry. 1999 Aug 10;38(32):10511-8. doi: 10.1021/bi9904712.
Aldehydes are known to inactivate cytochrome P450 in the reconstituted enzyme system containing NADPH and NADPH-cytochrome P450 reductase under aerobic conditions in a mechanism-based reaction involving heme adduct formation [Raner, G. M., Chiang, E. W. , Vaz, A. D. N., and Coon, M. J. (1997) Biochemistry 36, 4895-4902]. In the study presented here, artificial oxidants were used to examine the mechanism of aldehyde activation by purified P450 2B4 in the absence of the usual O(2)-reducing system, and the adducts that were formed were isolated and characterized. With hydrogen peroxide as the oxidant, 3-phenylpropionaldehyde gives an adduct with a mass corresponding to that of native heme modified by a phenylethyl group, presumably arising from the reaction of a peroxy-iron species with the aldehyde to give a peroxyhemiacetal, which upon deformylation yields the alkyl radical. NMR analysis indicated that the substitution is specifically at the gamma-meso position. In contrast, with m-chloroperbenzoic acid as the oxidant, an adduct is formed from 3-phenylpropionaldehyde with a mass that is consistent with the addition of a phenylpropionyl group, apparently arising by hydrogen abstraction from the aldehyde to give the carbonyl carbon radical. m-Chloroperbenzoic acid by itself forms a heme adduct with a mass corresponding to the addition of a chlorobenzoyloxy group apparently derived from homolytic oxygen-oxygen bond cleavage. These and other results with nonanal and 2-trans-nonenal support the concept that this versatile enzyme utilizes discrete oxidizing species in heme adduct formation from aldehydes.
已知在有氧条件下，醛类可在基于机制的反应中使含有NADPH和NADPH - 细胞色素P450还原酶的重组酶系统中的细胞色素P450失活，该反应涉及血红素加合物的形成[拉纳，G.M.，蒋，E.W.，瓦斯，A.D.N.，和库恩，M.J.（1997年）《生物化学》36，4895 - 4902]。在本文所述的研究中，使用人工氧化剂在不存在通常的O₂还原系统的情况下，研究纯化的P450 2B4激活醛类的机制，并对形成的加合物进行分离和表征。以过氧化氢为氧化剂时，3 - 苯基丙醛形成一种加合物，其质量与被苯乙基修饰的天然血红素的质量相对应，推测是由过氧铁物种与醛反应生成过氧半缩醛，该过氧半缩醛脱甲酰基后产生烷基自由基所致。核磁共振分析表明取代具体发生在γ - 中位。相比之下，以间氯过苯甲酸为氧化剂时，3 - 苯基丙醛形成一种加合物，其质量与添加苯丙酰基一致，显然是由醛上的氢原子被夺取生成羰基碳自由基所致。间氯过苯甲酸自身形成一种血红素加合物，其质量与添加一个显然源自氧 - 氧均裂的氯苯甲酰氧基相对应。这些以及关于壬醛和反 - 2 - 壬烯醛的其他结果支持了这样一种概念，即这种多功能酶在醛类形成血红素加合物的过程中利用不同的氧化物种。
