Contrasting Mechanisms of Aromatic and Aryl-Methyl Substituent Hydroxylation by the Rieske Monooxygenase Salicylate 5-Hydroxylase
Contrasting Mechanisms of Aromatic and Aryl-Methyl Substituent Hydroxylation by the Rieske Monooxygenase Salicylate 5-Hydroxylase
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DOI：
10.1021/acs.biochem.2c00610
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发表时间：
2022-12-30
期刊：
影响因子：
2.9
通讯作者：
Lipscomb，John D.
中科院分区：
文献类型：
--
作者：
Rogers，Melanie S.;Gordon，Adrian M.;Lipscomb，John D.
水杨酸-5-羟化酶的羟化酶组分（S5 HH）催化水杨酸的C5环羟基化，但当存在C5甲基取代基时切换为甲基羟基化。18 O2的使用揭示了芳香族和芳甲基羟基化都是由单加氧酶化学引起的。S5 HH的功能单元包括一个非血红素Fe（II）位点，其位于单电子还原的Rieske型铁硫簇的亚基边界的12处。过去的研究确定，底物结合附近的Fe（II），其次是O2结合到铁启动催化。停流单周转反应（STO）表明，Rieske簇转移电子的铁网站在催化过程中。它在这里示出，氟环取代基降低Rieske电子转移的速率常数，这意味着一个先前的反应的Fe（III）-superoxo中间体与基板。我们建议，铁成为完全氧化的Fe（III）-过氧基自由基中间体，允许Rieske电子转移发生。使用5-CD 3-水杨酸-d8的STO发生逆动力学同位素效应（KIE）。相比之下，未标记和5-CD 3-水杨酸-d8的1：1混合物的STO产生正常产物同位素效应。有人提出，芳香族和芳基甲基羟基化反应都是从与环碳的Fe（III）-超氧反应开始开始的，由于sp2→ sp3碳杂交而产生逆KIE。在Rieske电子转移后，所得的Fe（III）-过氧水杨酸中间体可以继续进行芳香族羟基化，而等价的芳基-甲基中间体的形成必须是可逆的，以允许产生正常产物同位素效应所需的底物交换。所得的Fe（III）-（氢）过氧中间体可以是反应性的或通过高价铁中间体来完成芳基-甲基羟基化。
The hydroxylase component (S5HH) of salicylate-5-hydroxylase catalyzes C5 ring hydroxylation of salicylate but switches to methyl hydroxylation when a C5 methyl substituent is present. The use of18O2reveals that both aromatic and aryl-methyl hydroxylations result from monooxygenase chemistry. The functional unit of S5HH comprises a nonheme Fe(II) site located 12 Å across a subunit boundary from a one-electron reduced Rieske-type iron–sulfur cluster. Past studies determined that substrates bind near the Fe(II), followed by O2binding to the iron to initiate catalysis. Stopped-flow-single-turnover reactions (STOs) demonstrated that the Rieske cluster transfers an electron to the iron site during catalysis. It is shown here that fluorine ring substituents decrease the rate constant for Rieske electron transfer, implying a prior reaction of an Fe(III)-superoxo intermediate with a substrate. We propose that the iron becomes fully oxidized in the resulting Fe(III)-peroxo-substrate-radical intermediate, allowing Rieske electron transfer to occur. STO using 5-CD3-salicylate-d8occurs with an inverse kinetic isotope effect (KIE). In contrast, STO of a 1:1 mixture of unlabeled and 5-CD3-salicylate-d8yields a normal product isotope effect. It is proposed that aromatic and aryl-methyl hydroxylation reactions both begin with the Fe(III)-superoxo reaction with a ring carbon, yielding the inverse KIE due to sp2→ sp3carbon hybridization. After Rieske electron transfer, the resulting Fe(III)-peroxo-salicylate intermediate can continue to aromatic hydroxylation, whereas the equivalent aryl-methyl intermediate formation must be reversible to allow the substrate exchange necessary to yield a normal product isotope effect. The resulting Fe(III)-(hydro)peroxo intermediate may be reactive or evolve through a high-valent iron intermediate to complete the aryl-methyl hydroxylation.