Tschirret-Guth R A, Ortiz de Montellano P R
Department of Pharmaceutical Chemistry, School of Pharmacy, University of California, San Francisco 94143-0446, USA.
Arch Biochem Biophys. 1996 Nov 1;335(1):93-101. doi: 10.1006/abbi.1996.0485.
Two mechanisms have been identified for the H2O2-dependent epoxidation of styrenes by sperm whale myoglobin (Mb) [S. Rao, A. Wilks, and P. R. Ortiz de Montellano, J. Biol. Chem. 268, 803-908 (1993)]: (a) ferryl (FeIV = O) oxygen transfer with retention of stereochemistry and incorporation of an oxygen from H2O2, and (b) protein peroxy radical cooxidation with loss of stereochemistry and incorporation of an oxygen from O2. As shown here, cis-beta-methylstyrene is preferentially oxidized to the trans-epoxide when the H2O2:Mb ratio is <0.5 but increasingly to the cis-isomer as the ratio increases to and above 1. At a high (4:1) H2O2:Mb ratio, both the absolute yield and the cis:trans-epoxide ratio increase in proportion to the cis-beta-methylstyrene concentration. A protein radical formed in the Mb-H2O2 reaction also causes dimer and trimer formation, maximum dimer formation (approximately 30%) being obtained with 1 equivalent of H2O2. At low H2O2:Mb ratios, the oxidation equivalents utilized for protein oligomerization and styrene oxidation account for the available H2O2. Previous studies have shown that His-64 is important for protein-mediated olefin cooxidation and Tyr-151/Tyr-103 for Mb dimerization. The W7F, W14F, and W7F/W14F Mb mutants have now been prepared and the W14F, but not W7F, mutation shown to modestly decrease cooxidation of cis-beta-methylstyrene to the trans-epoxide. Neither tryptophan mutation alters dimer formation. Dimer formation is modestly increased rather than decreased by styrene, suggesting that styrene cooxidation and dimerization do not compete. The results indicate that (a) cis-beta-methylstyrene cooxidation and protein dimerization, both of which are mediated by protein radicals, are favored at low H2O2:Mb ratios, (b) as the H2O2:Mb ratio increases, the ferryl epoxidation pathway surpasses the cooxidation mechanism, (c) Trp-14 but not Trp-7 influences olefin cooxidation, and (d) different, possibly nonequilibrating, radicals mediate olefin cooxidation and protein dimerization.
抹香鲸肌红蛋白（Mb）催化苯乙烯H2O2依赖性环氧化反应的机制已被确定为两种 [S. Rao、A. Wilks和P. R. Ortiz de Montellano，《生物化学杂志》268, 803 - 908 (1993)]：（a）铁（IV）=氧（ferryl）的氧转移，立体化学保持不变，且从H2O2中掺入一个氧原子；（b）蛋白质过氧自由基共氧化，立体化学消失，并从O2中掺入一个氧原子。如下所示，当H2O2:Mb比例<0.5时，顺式-β-甲基苯乙烯优先被氧化为反式环氧化物，但随着该比例增加到1及以上，越来越多地生成顺式异构体。在高（4:1）H2O2:Mb比例下，绝对产率和顺式:反式环氧化物比例均与顺式-β-甲基苯乙烯浓度成比例增加。Mb - H2O2反应中形成的蛋白质自由基还会导致二聚体和三聚体的形成，1当量的H2O2可实现最大程度的二聚体形成（约30%）。在低H2O2:Mb比例下，用于蛋白质寡聚化和苯乙烯氧化的氧化当量占可用H2O2的比例。先前的研究表明，His - 64对蛋白质介导的烯烃共氧化很重要，而Tyr - 151/Tyr - 103对Mb二聚化很重要。现已制备了W7F、W14F和W7F/W14F Mb突变体，结果表明W14F突变（而非W7F突变）会适度降低顺式-β-甲基苯乙烯向反式环氧化物的共氧化。两种色氨酸突变均未改变二聚体的形成。苯乙烯会适度增加而非减少二聚体的形成，这表明苯乙烯共氧化和二聚化并不相互竞争。结果表明：（a）顺式-β-甲基苯乙烯共氧化和蛋白质二聚化在低H2O2:Mb比例下均受青睐，二者均由蛋白质自由基介导；（b）随着H2O2:Mb比例增加，铁（IV）=氧环氧化途径超过共氧化机制；（c）Trp - 14而非Trp - 7影响烯烃共氧化；（d）不同的、可能非平衡的自由基介导烯烃共氧化和蛋白质二聚化。
