Evidence that dioxygen and substrate activation are tightly coupled in dopamine β-monooxygenase -: Implications for the reactive oxygen species
Evidence that dioxygen and substrate activation are tightly coupled in dopamine β-monooxygenase -: Implications for the reactive oxygen species
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DOI：
10.1074/jbc.m300797200
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发表时间：
2003-12-12
影响因子：
4.8
通讯作者：
Klinman, JP
中科院分区：
文献类型：
--
作者：
Evans, JP;Ahn, K;Klinman, JP
氧活化发生在各种各样的酶活性位点。先前提出的铜单加氧酶，多巴胺β-单加氧酶（DbetaM）的机制，涉及积累的活性氧中间体的性质的铜过氧或铜氧物种底物活化前。这些都让人想起细胞色素P-450的机制，其中血红素铁稳定活化的O-2物种。在此，我们报告了两个实验探测的活性氧物种在D β M。首先，我们已经合成了底物类似物，β，β-二氟苯乙胺，并研究其能力，诱导再氧化的预还原铜网站DbetaM混合后，与O-2在快速冷冻淬火条件下。与具有C-H活性键的基底相比，该实验未能产生EPR可检测的铜物质。这表明在O-2存在下酶结合铜位点的再氧化与C-H活化紧密相关，或者抗磁性物种Cu（II）- O-2（.）已经形成。在同源肽基-甘氨酸-α-羟基化单加氧酶的开放和完全溶剂可及的活性位点的背景下，并通过类比细胞色素P-450，在C-H裂解之前D β M中还原和活化的氧物质的积累预计会产生氧和底物消耗的一些解偶联。因此，我们已经研究了在何种程度上O-2和底物消耗耦合在D β M使用终点和初始速率实验协议。与基板的速率相差超过三个数量级，我们未能检测到任何解偶联的O-2吸收从产品形成。我们的结论是，有没有积累的活化形式的O-2前C-H抽象在D β M和肽基甘氨酸-α-羟基化单加氧酶类的铜单加氧酶，提出了一种机制，其中一个抗磁性的铜（II）- superoxo复合物，最初形成在非常低的水平，抽象的氢原子从基板上产生铜（II）-氢过氧和基板自由基作为中间体。随后每个亚基的第二个铜位点的参与完成反应循环，产生羟基化产物和水。
Oxygen activation occurs at a wide variety of enzyme active sites. Mechanisms previously proposed for the copper monooxygenase, dopamine beta-monooxygenase (DbetaM), involve the accumulation of an activated oxygen intermediate with the properties of a copper-peroxo or copper-oxo species before substrate activation. These are reminiscent of the mechanism of cytochrome P-450, where a heme iron stabilizes the activated O-2 species. Herein, we report two experimental probes of the activated oxygen species in DbetaM. First, we have synthesized the substrate analog, beta,beta-difluorophenethylamine, and examined its capacity to induce reoxidation of the prereduced copper sites of DbetaM upon mixing with O-2 under rapid freeze-quench conditions. This experiment fails to give rise to an EPR-detectable copper species, in contrast to a substrate with a C-H active bond. This indicates either that the reoxidation of the enzyme-bound copper sites in the presence of O-2 is tightly linked to C-H activation or that a diamagnetic species Cu(II) - O-2(.) has been formed. In the context of the open and fully solvent-accessible active site for the homologous peptidyl-glycine-alpha-hydroxylating monooxygenase and by analogy to cytochrome P-450, the accumulation of a reduced and activated oxygen species in DbetaM before C-H cleavage would be expected to give some uncoupling of oxygen and substrate consumption. We have, therefore, examined the degree to which O-2 and substrate consumption are coupled in DbetaM using both end point and initial rate experimental protocols. With substrates that differ by more than three orders of magnitude in rate, we fail to detect any uncoupling of O-2 uptake from product formation. We conclude that there is no accumulation of an activated form of O-2 before C-H abstraction in the DbetaM and peptidylglycine-alpha-hydroxylating monooxygenase class of copper monooxygenases, presenting a mechanism in which a diamagnetic Cu( II)- superoxo complex, formed initially at very low levels, abstracts a hydrogen atom from substrate to generate Cu( II)- hydroperoxo and substrate-free radical as intermediates. Subsequent participation of the second copper site per subunit completes the reaction cycle, generating hydroxylated product and water.