Miller V P, DePillis G D, Ferrer J C, Mauk A G, Ortiz de Montellano P R
Department of Pharmaceutical Chemistry, School of Pharmacy, University of California, San Francisco 94143-0446.
J Biol Chem. 1992 May 5;267(13):8936-42.
Recombinant cytochrome c peroxidase (CcP) and a W51A mutant of CcP, in contrast to other classical peroxidases, react with phenylhydrazine to give sigma-bonded phenyl-iron complexes. The conclusion that the heme iron is accessible to substrates is supported by the observation that CcP and W51A CcP oxidize thioanisole to the racemic sulfoxide with quantitative incorporation of oxygen from H2O2. Definitive evidence for an open active site is provided by stereoselective epoxidation by both enzymes of styrene, cis-beta-methylstyrene, and trans-beta-methylstyrene. trans-beta-methylstyrene yields exclusively the trans-epoxide, but styrene yields the epoxide and phenylacetaldehyde, and cis-beta-methylstyrene yields both the cis- and trans-epoxides and 1-phenyl-2-propanone. The sulfoxide, stereoretentive epoxides, and 1-phenyl-2-propanone are formed by ferryl oxygen transfer mechanisms because their oxygen atom derives from H2O2. In contrast, the oxygen in the trans-epoxide from the cis-olefin derives primarily from molecular oxygen and is probably introduced by a protein cooxidation mechanism. cis-[1,2-2H]-1-Phenyl-1-propene is oxidized to [1,1-2H]-1-phenyl-2-propanone without a detectable isotope effect on the epoxide:ketone product ratio. The phenyl-iron complex is not formed and substrate oxidation is not observed when the prosthetic group is replaced by delta-meso-ethylheme. CcP thus has a sufficiently open active site to form a phenyl-iron complex, to oxidize thioanisole to the sulfoxide, and to epoxidize styrene and beta-methylstyrene. The results indicate that a ferryl (Fe(IV) = O)/protein radical pair can be coupled to achieve two-electron oxidations. The unique ability of CcP to catalyze monooxygenation reactions does not conflict with its peroxidase function because cytochrome c is oxidized at a distinct surface site (DePillis, G. D., Sishta, B. P., Mauk, A. G., and Ortiz de Montellano, P. R. (1991) J. Biol. Chem. 266, 19334-19341).
与其他经典过氧化物酶不同，重组细胞色素c过氧化物酶（CcP）及其W51A突变体与苯肼反应生成σ键合的苯基铁配合物。CcP和W51A CcP能将苯甲硫醚氧化为外消旋亚砜，并定量地从H2O2中引入氧，这一观察结果支持了血红素铁可被底物接近的结论。两种酶对苯乙烯、顺式-β-甲基苯乙烯和反式-β-甲基苯乙烯进行立体选择性环氧化，为开放活性位点提供了确凿证据。反式-β-甲基苯乙烯仅生成反式环氧化物，但苯乙烯生成环氧化物和苯乙醛，顺式-β-甲基苯乙烯生成顺式和反式环氧化物以及1-苯基-2-丙酮。亚砜、立体保持性环氧化物和1-苯基-2-丙酮是通过高铁氧转移机制形成的，因为它们的氧原子来自H2O2。相比之下，顺式烯烃生成的反式环氧化物中的氧主要来自分子氧，可能是通过蛋白质共氧化机制引入的。顺式-[1,2-2H]-1-苯基-1-丙烯被氧化为[1,1-2H]-1-苯基-2-丙酮，环氧化物：酮产物比例未观察到可检测的同位素效应。当辅基被δ-内消旋-乙基血红素取代时，不形成苯基铁配合物，也未观察到底物氧化。因此，CcP具有足够开放的活性位点以形成苯基铁配合物，将苯甲硫醚氧化为亚砜，并将苯乙烯和β-甲基苯乙烯环氧化。结果表明，高铁（Fe(IV)=O）/蛋白质自由基对可以偶联以实现双电子氧化。CcP催化单加氧反应的独特能力与其过氧化物酶功能并不冲突，因为细胞色素c在一个独特的表面位点被氧化（DePillis, G. D., Sishta, B. P., Mauk, A. G., and Ortiz de Montellano, P. R. (1991) J. Biol. Chem. 266, 19334 - 19341）。
