Recent studies have revealed critical roles for the local environments surrounding metallocofactors, such as the newly identified CuD site in particulate methane monooxygenases (pMMOs) and the second sphere aromatic residues in lytic polysaccharide monooxygenases (LPMOs), implicated in the protection against oxidative damage. However, these features are subjects of continued debate. Our work utilizes biotin-streptavidin (Sav) technology to develop artificial metalloproteins (ArMs) that mimic the active sites of natural copper metalloenzymes. By engineering ArMs with aromatic residues within their secondary coordination spheres, we systematically investigate the influence of these residues on Cu reactivity and oxidant activation. We demonstrate that the placement and orientation of tyrosine relative to the Cu cofactor critically affect the oxidation outcomes upon exposure to hydrogen peroxide. A key finding is the interplay between the coordination of an active site asparagine and the incorporation of aromatic residues proximal to the artificial Cu cofactor, which are the only variants where oxidation of an engineered residues is observed. These findings underscore the importance of the secondary coordination sphere in modulating Cu center reactivity, suggest a role for amide coordination in C–H bond activation by pMMOs, and potential inactivation pathways in natural copper enzymes like LPMOs.

中文翻译：

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工程化 Cu 蛋白中活性位点芳香族残基的选择性氧化


最近的研究揭示了金属共生因子周围局部环境的关键作用，例如颗粒甲烷单加氧酶 （pMMO） 中新发现的 CuD 位点和裂解多糖单加氧酶 （LPMO） 中的第二球芳香族残基，与防止氧化损伤有关。然而，这些特征是持续争论的主题。我们的工作利用生物素-链霉亲和素 （Sav） 技术开发模拟天然铜金属酶活性位点的人工金属蛋白 （ArM）。通过在其次级配位球内使用芳香族残基对 ArM 进行工程改造，我们系统地研究了这些残基对 Cu 反应性和氧化剂活化的影响。我们证明，酪氨酸相对于 Cu 辅因子的位置和方向对暴露于过氧化氢后的氧化结果产生严重影响。一个关键的发现是活性位点天冬酰胺的配位与人工 Cu 辅因子近端芳香族残基的掺入之间的相互作用，这是观察到工程残基氧化的唯一变体。这些发现强调了次级配位球在调节 Cu 中心反应性中的重要性，表明酰胺配位在 pMMO 的 C-H 键激活中的作用，以及 LPMO 等天然铜酶中的潜在灭活途径。