Gan Yunqiao J, Hazel Joseph M, Searle Brian C, Shafaat Hannah S
Department of Chemistry and Biochemistry, The Ohio State University, 100 W 18th Avenue, Columbus, OH 43210, USA; Department of Chemistry and Biochemistry, University of California, Los Angeles, 607 Charles E. Young Drive East, Los Angeles, CA 90095, USA.
Department of Chemistry and Biochemistry, The Ohio State University, 100 W 18th Avenue, Columbus, OH 43210, USA.
J Inorg Biochem. 2025 Sep;270:112933. doi: 10.1016/j.jinorgbio.2025.112933. Epub 2025 Apr 23.
The R2-like ligand binding oxidase (R2lox) forms a novel tyrosine-valine crosslink upon O activation, reflecting an overall two-electron oxidation reaction. However, the mechanism through which the crosslink is formed is under debate, as the reaction can be initiated through either tyrosine OH or valine CH bond activation. Here, we utilized selective isotopic labeling and several spectroscopic techniques to probe the mechanism of this oxidation process. The results suggest that R2lox is capable of performing CH bond activation, with both solvent and CH kinetic isotope effects of approximately 2. Signatures of a high-valent Mn/Fe intermediate were observed through rapid-freeze-quench EPR that resemble an analogous intermediate found in the heterobimetallic radical-initiating enzyme, class Ic ribonucleotide reductase. This study provides a lower bound on the free energies of CH bonds that can be cleaved by Mn/Fe cofactors and suggests the potential for such enzymes to functionally replace Fe/Fe cofactors in a range of reactions.
R2样配体结合氧化酶（R2lox）在O激活时形成一种新型的酪氨酸-缬氨酸交联，这反映了一个整体的双电子氧化反应。然而，交联形成的机制仍存在争议，因为反应可以通过酪氨酸OH或缬氨酸CH键的激活来启动。在这里，我们利用选择性同位素标记和几种光谱技术来探究这种氧化过程的机制。结果表明，R2lox能够进行CH键的激活，溶剂和CH动力学同位素效应均约为2。通过快速冷冻淬灭EPR观察到高价Mn/Fe中间体的特征，类似于在异双金属自由基引发酶Ic类核糖核苷酸还原酶中发现的类似中间体。这项研究提供了可被Mn/Fe辅因子裂解的CH键自由能的下限，并表明这类酶在一系列反应中功能替代Fe/Fe辅因子的潜力。
