Cytochrome P450 OleT utilizes hydrogen peroxide (H₂O₂) to catalyze the decarboxylation or hydroxylation of fatty acid (FA) substrates. Both reactions are initiated through the abstraction of a substrate hydrogen atom by the high-valent iron–oxo intermediate known as Compound I. Here, we specifically probe the influence of substrate coordination on OleT reaction partitioning through the combined use of fluorescent and electron paramagnetic resonance (EPR)-active FA probes and mutagenesis of a structurally disordered F–G loop that is distal from the heme-iron active site. Both probes are efficiently metabolized by OleT and efficiently trigger the formation of Compound I. Transient fluorescence and EPR reveal a slow product release step, mediated by the F–G loop, that limits OleT turnover. A single-amino acid change or excision of the loop reveals that this region establishes critical interactions to anchor FA substrates in place. The stabilization afforded by the F–G loop is essential for regulating regiospecific C–H abstraction and allowing for efficient decarboxylation to occur. These results highlight a regulatory strategy whereby the fate of activated oxygen species can be controlled at distances far removed from the site of chemistry.

中文翻译：

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远端环控制细胞色素P450脱羧酶中的产品释放以及化学和区域选择性

细胞色素P450 OleT利用过氧化氢（H ₂ O ₂）以催化脂肪酸（FA）底物的脱羧或羟基化。这两个反应都是通过高价铁-氧代中间体（称为化合物I）提取底物氢原子而引发的。在这里，我们通过荧光和电子顺磁共振的结合使用，专门探讨底物配位对OleT反应分配的影响。 （EPR）活性FA探针和结构异常的F–G环的诱变作用，该环位于血红素铁活性位点的远端。两种探针都可以通过OleT有效代谢，并有效触发化合物I的形成。瞬态荧光和EPR揭示了由F–G环介导的缓慢的产物释放步骤，这限制了OleT的转化。单个氨基酸的变化或环的切除揭示了该区域建立了关键的相互作用，从而将FA底物锚定在适当的位置。F–G环提供的稳定作用对于调节区域特异性C–H提取并允许有效的脱羧作用至关重要。这些结果突显了一种调节策略，通过该策略可以将活性氧物种的命运控制在远离化学位点的距离处。