Prakash Divyansh, Wu Yao, Misra Sandeep K, Sampath Nishanthini, Wang Binju, Chakraborty Saumen
Department of Chemistry and Biochemistry, University of Mississippi, University, MS, 38677, USA.
State Key Laboratory of Physical Chemistry of Solid Surfaces and Fujian Provincial Key Laboratory of Theoretical and Computational Chemistry, College of Chemistry and Chemical Engineering, Xiamen University, Xiamen, P. R. China.
Chemistry. 2025 Sep 16;31(52):e01245. doi: 10.1002/chem.202501245. Epub 2025 Aug 13.
De novo-designed artificial Cu protein (ArCuP), 3SCC, featuring a trigonal Cu(His) binding environment, activates HO, O, and benzylic C─H bonds of abiotic substrates. We outline the mechanism of ArCuP-catalyzed C─H peroxidation of one such abiotic substrate, the peroxidation of benzyl alcohol (BA) to benzaldehyde. The Cu(HO) complex undergoes homolytic cleavage, producing Cu-OH and OH, akin to the lytic polysaccharide monooxygenases (LPMOs). The rate-limiting step is found in a PCET process, where the Cu-OH species accepts the benzylic C─H proton, accompanied by electron transfer from the O-centered substrate radical, to produce benzaldehyde. The C─H peroxidation is modulated by outer-sphere modifications, with the I12A-3SCC variant having the highest catalytic proficiency. Combined OH monitoring and proteomics analysis reveal that the I12A variant produces the least amount of OH, indicating no oxidation of the active site His residues, unlike other ArCuPs. The chemical and thermal stability of the I12A variant contributes to its superior reactivity. The presence of substrate significantly lowers protein-level oxidation, similar to LPMOs and other Cu and heme-based enzymes.
具有三角铜（组氨酸）结合环境的从头设计人工铜蛋白（ArCuP）3SCC可激活非生物底物的羟基、氧和苄基碳氢键。我们概述了ArCuP催化一种此类非生物底物苄醇（BA）过氧化生成苯甲醛的碳氢键过氧化机制。铜（羟基）配合物发生均裂，生成铜-羟基和羟基，这类似于裂解多糖单加氧酶（LPMO）。限速步骤存在于一个质子耦合电子转移（PCET）过程中，其中铜-羟基物种接受苄基碳氢键质子，同时伴随着来自以氧为中心的底物自由基的电子转移，生成苯甲醛。碳氢键过氧化可通过外层修饰进行调节，I12A-3SCC变体具有最高的催化效率。结合羟基监测和蛋白质组学分析表明，与其他ArCuP不同，I12A变体产生的羟基量最少，这表明活性位点组氨酸残基未被氧化。I12A变体的化学和热稳定性有助于其卓越的反应活性。底物的存在显著降低了蛋白质水平的氧化，这与LPMO以及其他基于铜和血红素的酶类似。
