Peroxo-iron and oxenoid-iron species as alternative oxygenating agents in cytochrome P450-catalyzed reactions: switching by threonine-302 to alanine mutagenesis of cytochrome P450 2B4.
Peroxo-iron and oxenoid-iron species as alternative oxygenating agents in cytochrome P450-catalyzed reactions: switching by threonine-302 to alanine mutagenesis of cytochrome P450 2B4.
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DOI：
10.1073/pnas.93.10.4644
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发表时间：
1996-05
影响因子：
11.1
通讯作者：
Alfin D. N. Vaz;S. Pernecky;G. Raner;M. Coon
中科院分区：
文献类型：
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作者：
Alfin D. N. Vaz;S. Pernecky;G. Raner;M. Coon
在生物催化剂中，细胞色素P450在其异构体、诱导剂、底物和催化的化学反应类型的多样性方面是无与伦比的。在本研究中，给出的证据表明，这种多功能性延伸到活性氧化剂的性质。尽管来自几个实验室的机械证据指出P450催化的氧化反应中的高价铁-类氧物种，但Akhtar及其同事[Akhtar，M.，Calder，M. R.，Corina，D. L. & Wright，J. N.（1982）Biochem.J.201，569-580]提出，在P450芳香酶作用下的类固醇脱乙酰化中涉及铁-过氧物质。最近，我们已经表明，纯化的肝微粒体P450细胞色素，包括苯巴比妥诱导的P450 2B 4，催化类似脱醛的一系列异生醛与烯烃的形成。本文研究了苏氨酸302定点突变为丙氨酸对N-端氨基酸2-27缺失的重组P450 2B 4活性的影响[2B 4（δ 2 -27）]利用来自其他实验室的证据，即细菌P450中相应的突变通过阻断质子传递到细胞中，干扰了氧分子对氧杂环物种的激活。活性部位用P450 2B 4、2B 4（delta 2 -27）和2B 4（delta 2 -27）T302 A测定了NADPH氧化、过氧化氢产生和四种底物（包括苄非他明N-脱甲基化产生的甲醛、1-苯基乙醇氧化产生的苯乙酮、环己烷羟基化产生的环己醇和环己烷甲醛脱甲基产生的环己烯）的产物形成速率。替换的苏氨酸残基的截短的细胞色素得到了1.6至2.5倍的增加，在过氧化物的形成在底物的存在下，但导致减少产品的形成，从苄非他明（9倍），环己烷（4倍），和1-苯基乙醇（2倍）。与此形成鲜明对比的是，T302 A突变体对环己烷甲醛的脱乙酰化增加了约10倍。基于这些发现和我们先前的证据，即醛脱乙酰基是由添加的H2 O2支持的，而不是由人工氧化剂支持的，我们得出结论，铁过氧物质是直接的氧供体。还有待确定的是，涉及P450的许多其他氧化反应中的哪一个利用了该物种，以及过氧铁和类氧合铁在多大程度上与该多功能催化剂的许多异构体一起用作替代氧化剂。
Among biological catalysts, cytochrome P450 is unmatched in its multiplicity of isoforms, inducers, substrates, and types of chemical reactions catalyzed. In the present study, evidence is given that this versatility extends to the nature of the active oxidant. Although mechanistic evidence from several laboratories points to a hypervalent iron-oxenoid species in P450-catalyzed oxygenation reactions, Akhtar and colleagues [Akhtar, M., Calder, M. R., Corina, D. L. & Wright, J. N. (1982) Biochem. J. 201, 569-580] proposed that in steroid deformylation effected by P450 aromatase an iron-peroxo species is involved. We have shown more recently that purified liver microsomal P450 cytochromes, including phenobarbital-induced P450 2B4, catalyze the analogous deformylation of a series of xenobiotic aldehydes with olefin formation. The investigation presented here on the effect of site-directed mutagenesis of threonine-302 to alanine on the activities of recombinant P450 2B4 with N-terminal amino acids 2-27 deleted [2B4 (delta2-27)] makes use of evidence from other laboratories that the corresponding mutation in bacterial P450s interferes with the activation of dioxygen to the oxenoid species by blocking proton delivery to the active site. The rates of NADPH oxidation, hydrogen peroxide production, and product formation from four substrates, including formaldehyde from benzphetamine N-demethylation, acetophenone from 1-phenylethanol oxidation, cyclohexanol from cyclohexane hydroxylation, and cyclohexene from cyclohexane carboxaldehyde deformylation, were determined with P450s 2B4, 2B4 (delta2-27), and 2B4 (delta2-27) T302A. Replacement of the threonine residue in the truncated cytochrome gave a 1.6- to 2.5-fold increase in peroxide formation in the presence of a substrate, but resulted in decreased product formation from benzphetamine (9-fold), cyclohexane (4-fold), and 1-phenylethanol (2-fold). In sharp contrast, the deformylation of cyclohexane carboxaldehyde by the T302A mutant was increased about 10-fold. On the basis of these findings and our previous evidence that aldehyde deformylation is supported by added H202, but not by artificial oxidants, we conclude that the iron-peroxy species is the direct oxygen donor. It remains to be established which of the many other oxidative reactions involving P450 utilize this species and the extent to which peroxo-iron and oxenoid-iron function as alternative oxygenating agents with the numerous isoforms of this versatile catalyst.