A proton-shuttle mechanism mediated by the porphyrin in benzene hydroxylation by cytochrome P450 enzymes
A proton-shuttle mechanism mediated by the porphyrin in benzene hydroxylation by cytochrome P450 enzymes
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DOI：
10.1021/ja034142f
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发表时间：
2003-06-18
影响因子：
15
通讯作者：
Shaik, S
中科院分区：
文献类型：
--
作者：
de Visser, SP;Shaik, S
苯羟基化是化学催化中的一个基本过程。在自然界中，该反应是由细胞色素 P450 酶通过氧转移催化的，其机制仍然存在争议，相当复杂。该论文使用混合密度泛函计算来阐明苯通过酶的活性物质（高价铁氧卟啉物质）转化为苯酚、氧化苯和酮的机制。与之前一样，模拟了蛋白质极性和氢键对活性物种的影响（Ogliaro, F.；Cohen, S.；de Visser, S. P.；Shaik, S. J. Am. Chem. Soc. 2000, 122, 12892-12893）。经证实，反应不会通过夺氢或初始电子转移进行（Ortiz de Montellano, P. R. In Cytochrome P450: Structure, Mechanism and Biochemistry, 2nd ed.；Ortiz de Montellano, P. R., Ed.；Plenum Press：New York，1995；第 8 章，第 245-303 页）。根据最新的实验结论，理论计算表明反应性是亲电子和自由基途径的相互作用，涉及对苯的 pi 系统的初始攻击以产生 α 配合物 (Korzekwa, K. R.; Swinney, D. C.; Trager, W. T. Biochemistry 1989, 28, 9019-9027)。主要反应通道是亲电子的，并通过阳离子西格玛络合物 (2)3 进行，该络合物涉及由阳离子苯部分和阴离子铁卟啉组成的内部离子对。小通道通过自由基σ复合物(2)2的中间作用进行，其中苯部分是自由基，铁卟啉部分是中性的。这些中间体闭环产生苯氧化物产物 ((2)4)，它不会重排为苯酚 ((2)7) 或环己烯酮 ((2)6)。虽然这种重排可以在生理条件下通过酸催化在酶促后发生，但计算揭示了一种新的机制，即酶的活性物质直接催化苯酚和环己烯酮的产生。这种酶促机制涉及由卟啉环通过 N-质子化中间体 (2)5 介导的质子穿梭，该中间体将质子传递至氧原子以形成苯酚 ((2)7) 或传递至邻碳原子以产生环己烯酮产物 ((2)6)。通过这种质子穿梭机制形成苯酚将与通过外部酸催化将苯氧化物非酶促转化为苯酚竞争。假设 (2)5 没有完全热化，这种新颖的机制也可以解释由于氢从羟基化位点迁移到相邻碳（所谓的“NIH 位移”（Jerina, D. M.; Daly, J. W. Science 1974, 185, 573-582）），活化的 C-H 键的原始氢存在部分骨架保留。因此，一般来说，计算发现的卟啉质子穿梭机制表明，除了通过将芳烃氧化物转化为苯酚和酮来非酶促生产这些物质之外，还存在一种将苯直接转化为苯酚和酮的酶促途径。根据在一些烯烃环氧化反应期间观察到的H/D交换，讨论了P450化学中质子化卟啉中间体的潜在普遍性（Groves，J. T.；Avaria-Neisser，G. M.；Imachi，M.；Kuczkowski，R. J. Am. Soc. 1986，108，3837-3838）和血红素烷基化产物的一般观察（Kunze，K. L.；Mangold，B. L. K.；Wheeler，C.；O rtiz）。德蒙特拉诺，P.R.J. 化学。1983 年，258，4202-4207。苯氧化与苯氧化之间的竞争、相似点和差异。讨论了烯烃环氧化和烷烃基C-H羟基化反应，并与相关实验和计算数据进行了比较。苯羟基化中低自旋反应性的主导地位即。烯烃环氧化和烷烃羟基化中的二态反应性（Shaik, S.；de Visser, S. P.；Ogliaro, F.；Schwarz, H.；Schroder, D. Curr. Opin. Chem. Biol. 2002, 6, 556-56）可追溯到键活化步骤中苯共振能的损失。
Benzene hydroxylation is a fundamental process in chemical catalysis. In nature, this reaction is catalyzed by the enzyme cytochrome P450 via oxygen transfer in a still debated mechanism of considerable complexity. The paper uses hybrid density functional calculations to elucidate the mechanisms by which benzene is converted to phenol, benzene oxide, and ketone, by the active species of the enzyme, the high-valent iron-oxo porphyrin species. The effects of the protein polarity and hydrogen-bonding donation to the active species are mimicked, as before (Ogliaro, F.; Cohen, S.; de Visser, S. P.; Shaik, S. J. Am. Chem. Soc. 2000, 122, 12892-12893). It is verified that the reaction does not proceed either by hydrogen abstraction or by initial electron transfer (Ortiz de Montellano, P. R. In Cytochrome P450: Structure, Mechanism and Biochemistry, 2nd ed.; Ortiz de Montellano, P. R., Ed.; Plenum Press: New York, 1995; Chapter 8, pp 245-303). In accord with the latest experimental conclusions, the theoretical calculations show that the reactivity is an interplay of electrophilic and radicalar pathways, which involve an initial attack on the pi-system of the benzene to produce a-complexes (Korzekwa, K. R.; Swinney, D. C.; Trager, W. T. Biochemistry 1989, 28, 9019-9027). The dominant reaction channel is electrophilic and proceeds via the cationic sigma-complex, (2)3, that involves an internal ion pair made from a cationic benzene moiety and an anionic iron porphyrin. The minor channel proceeds by intermediacy of the radical sigma-complex, (2)2, in which the benzene moiety is radicalar and the iron-porphyrin moiety is neutral. Ring closure in these intermediates produces the benzene oxide product ((2)4), which does not rearrange to phenol ((2)7) or cyclohexenone ((2)6). While such a rearrangement can occur post-enzymatically under physiological conditions by acid catalysis, the computations reveal a novel mechanism whereby the active species of the enzyme catalyzes directly the production of phenol and cyclohexenone. This enzymatic mechanism involves proton shuttles mediated by the porphyrin ring through the N-protonated intermediate, (2)5, which relays the proton either to the oxygen atom to form phenol ((2)7) or to the ortho-carbon atom to produce cyclohexenone product ((2)6). The formation of the phenol via this proton-shuttle mechanism will be competitive with the nonenzymatic conversion of benzene oxide to phenol by external acid catalysis. With the assumption that (2)5 is not fully thermalized, this novel mechanism would account also for the observation that there is a partial skeletal retention of the original hydrogen of the activated C-H bond, due to migration of the hydrogen from the site of hydroxylation to the adjacent carbon (so-called "NIH shift" (Jerina, D. M.; Daly, J. W. Science 1974, 185, 573-582)). Thus, in general, the computationally discovered mechanism of a porphyrin proton shuttle suggests that there is an enzymatic pathway that converts benzene directly to a phenol and ketone, in addition to nonenzymatic production of these species by conversion of arene oxide to phenol and ketone.The potential generality of protonated porphyrin intermediates in P450 chemistry is discussed in the light of the H/D exchange observed during some olefin epoxidation reactions (Groves, J. T.; Avaria-Neisser, G. E.; Fish, K. M.; Imachi, M.; Kuczkowski, R. J. Am. Chem. Soc. 1986, 108, 3837-3838) and the general observation of heme alkylation products (Kunze, K. L.; Mangold, B. L. K.; Wheeler, C.; Beilan, H. S.; O rtiz de Montellano, P.R.J. Biol. Chem. 1983, 258, 4202-4207). The competition, similarities, and differences between benzene oxidation viz. olefin epoxidation and alkanyl C-H hydroxylation are discussed, and comparison is made with relevant experimental and computational data. The dominance of low-spin reactivity in benzene hydroxylation viz. two-state reactivity (Shaik, S.; de Visser, S. P.; Ogliaro, F.; Schwarz, H.; Schroder, D. Curr. Opin. Chem. Biol. 2002, 6, 556-56) in olefin epoxidation and alkane hydroxylation is traced to the loss of benzene resonance energy during the bond activation step.