Salicylate 5-Hydroxylase: Intermediates in Aromatic Hydroxylation by a Rieske Monooxygenase.
Salicylate 5-Hydroxylase: Intermediates in Aromatic Hydroxylation by a Rieske Monooxygenase.
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DOI：
10.1021/acs.biochem.9b00292
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发表时间：
2019-05
期刊：
影响因子：
2.9
通讯作者：
M. Rogers;J. Lipscomb
中科院分区：
文献类型：
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作者：
M. Rogers;J. Lipscomb
Rieske加氧酶（ROs）催化大量的氧化化学反应。我们已经证明，顺式二氢二醇形成Rieske双加氧酶首先通过活性位点非血红素Fe(III)-超氧化物与其芳香底物反应；来自Rieske团簇的电子转移完成了生成产物的反应。或者，两电子还原的Fe(III)-过氧或羟基-Fe(V)-氧活性氧中间体是可能的，并且可以被其他ROs利用来扩大催化范围。本文研究了不形成顺式二氢二醇的Rieske单加氧酶（水杨酸5-羟化酶）的反应。单次转化动力学研究表明水杨酸盐与O2的结合速度很快。形成gentisate产物所需的Rieske电子的转移通过~ 12 Å以上的键发生，并且也必须非常快。然而，观察到的反应速率常数比预期的要慢得多，并且对底物类型很敏感。这表明，与水杨酸盐的初始反应与Rieske双加氧酶使用相同的Fe(III)-超氧中间体发生，并且该反应限制了观察到的电子转移速率。产物在活性位点形成后，在g = 4.3处观察到一个瞬态中间体（λmax = 700 nm）和电子顺磁共振（EPR）。17O2 （I = 5/2）的使用导致g = 4.3信号的超细增宽，表明龙胆酸通过中间体中的C5-OH与单核铁结合。生色团和EPR信号允许研究催化循环中的产物释放。单次和多次转化反应的动力学比较表明，金属中心的再还原加速了产物释放约300倍，为ROs的调节机制提供了新的见解。
Rieske oxygenases (ROs) catalyze a large range of oxidative chemistry. We have shown that cis-dihydrodiol-forming Rieske dioxygenases first react with their aromatic substrates via an active site nonheme Fe(III)-superoxide; electron transfer from the Rieske cluster then completes the product-forming reaction. Alternatively, two-electron-reduced Fe(III)-peroxo or hydroxo-Fe(V)-oxo activated oxygen intermediates are possible and may be utilized by other ROs to expand the catalytic range. Here, the reaction of a Rieske monooxygenase, salicylate 5-hydroxylase, that does not form a cis-dihydrodiol is examined. Single-turnover kinetic studies show fast binding of salicylate and O2. Transfer of the Rieske electron required to form the gentisate product occurs through bonds over ∼12 Å and must also be very fast. However, the observed rate constant for this reaction is much slower than expected and sensitive to substrate type. This suggests that initial reaction with salicylate occurs using the same Fe(III)-superoxo-level intermediate as Rieske dioxygenases and that this reaction limits the observed rate of electron transfer. A transient intermediate (λmax = 700 nm) with an electron paramagnetic resonance (EPR) at g = 4.3 is observed after the product is formed in the active site. The use of 17O2 ( I = 5/2) results in hyperfine broadening of the g = 4.3 signal, showing that gentisate binds to the mononuclear iron via its C5-OH in the intermediate. The chromophore and EPR signal allow study of product release in the catalytic cycle. Comparison of the kinetics of single- and multiple-turnover reactions shows that re-reduction of the metal centers accelerates product release ∼300-fold, providing insight into the regulatory mechanism of ROs.