Evans John P, Ahn Kyunghye, Klinman Judith P
Department of Chemistry, University of California, Berkeley, California 94720-1460, USA.
J Biol Chem. 2003 Dec 12;278(50):49691-8. doi: 10.1074/jbc.M300797200. Epub 2003 Sep 9.
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 O2 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 O2 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 O2 is tightly linked to C-H activation or that a diamagnetic species Cu(II)-O2* has been formed. In the context of the open and fully solvent-accessible active site for the homologous peptidylglycine-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 O2 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 O2 uptake from product formation. We conclude that there is no accumulation of an activated form of O2 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.
氧的活化发生在多种酶的活性位点。先前提出的铜单加氧酶——多巴胺β-单加氧酶（DβM）的作用机制，涉及在底物活化之前积累具有铜过氧或铜氧物种性质的活性氧中间体。这些机制让人联想到细胞色素P-450的机制，其中血红素铁稳定活性O₂物种。在此，我们报告了两种探测DβM中活性氧物种的实验方法。首先，我们合成了底物类似物β,β-二氟苯乙胺，并在快速冷冻淬灭条件下与O₂混合时，检测其诱导DβM预还原铜位点再氧化的能力。与具有C-H活性键的底物相比，该实验未能产生可通过电子顺磁共振（EPR）检测到的铜物种。这表明要么在O₂存在下酶结合铜位点的再氧化与C-H活化紧密相关，要么形成了一种抗磁性物种Cu(II)-O₂*。在同源肽基甘氨酸α-羟化单加氧酶开放且完全可被溶剂接近的活性位点的背景下，并类比细胞色素P-450，预计在DβM中C-H裂解之前还原态和活性氧物种的积累会导致氧和底物消耗的某种解偶联。因此，我们使用终点法和初始速率实验方案研究了DβM中O₂和底物消耗的偶联程度。对于反应速率相差三个数量级以上的底物，我们未能检测到O₂摄取与产物形成之间的任何解偶联。我们得出结论，在DβM和肽基甘氨酸α-羟化单加氧酶类铜单加氧酶中，C-H提取之前不存在活性O₂形式的积累，提出了一种机制，即最初以非常低的水平形成的抗磁性Cu(II)-超氧配合物从底物中提取一个氢原子，生成Cu(II)-氢过氧物和底物自由基作为中间体。每个亚基的第二个铜位点随后参与完成反应循环，生成羟基化产物和水。
