The nonheme iron enzyme ScoE catalyzes the biosynthesis of an isonitrile substituent in a peptide chain. To understand details of the reaction mechanism we created a large active site cluster model of 212 atoms that contains substrate, the active oxidant and the first- and second-coordination sphere of the protein and solvent. Several possible reaction mechanisms were tested and it is shown that isonitrile can only be formed through two consecutive catalytic cycles that both use one molecule of dioxygen and α-ketoglutarate. In both cycles the active species is an iron(IV)-oxo species that in the first reaction cycle reacts through two consecutive hydrogen atom abstraction steps: first from the N–H group and thereafter from the C–H group to desaturate the NH-CH₂ bond. The alternative ordering of hydrogen atom abstraction steps was also tested but found to be higher in energy. Moreover, the electronic configurations along that pathway implicate an initial hydride transfer followed by proton transfer. We highlight an active site Lys residue that is shown to donate charge in the transition states and influences the relative barrier heights and bifurcation pathways. A second catalytic cycle of the reaction of iron(IV)-oxo with desaturated substrate starts with hydrogen atom abstraction followed by decarboxylation to give isonitrile directly. The catalytic cycle is completed with a proton transfer to iron(II)-hydroxo to generate the iron(II)-water resting state. The work is compared with experimental observation and previous computational studies on this system and put in a larger perspective of nonheme iron chemistry.

非血红素铁酶 ScoE 催化肽链中异腈取代基的生物合成。为了了解反应机制的细节，我们创建了一个包含 212 个原子的大型活性位点簇模型，其中包含底物、活性氧化剂以及蛋白质和溶剂的第一和第二配位球。测试了几种可能的反应机制，结果表明异腈只能通过两个连续的催化循环形成，这两个循环都使用一分子双氧和 α-酮戊二酸。在两个循环中，活性物质都是铁 (IV)-氧代物质，在第一个反应循环中通过两个连续的氢原子提取步骤反应：首先从 N-H 基团，然后从 C-H 基团去饱和 NH-通道₂键。还测试了氢原子提取步骤的替代顺序，但发现能量更高。此外，沿着该路径的电子配置暗示了最初的氢化物转移，然后是质子转移。我们强调了一个活性位点 Lys 残基，该残基显示出在过渡态中提供电荷并影响相对势垒高度和分叉途径。铁 (IV)-氧代与去饱和底物反应的第二个催化循环从氢原子提取开始，然后脱羧直接得到异腈。催化循环通过质子转移到铁 (II)-羟基以生成铁 (II)-水静止状态而完成。