Aix Marseille Université, CNRS, Centrale Marseille, iSm2, Marseille 13397, France.
Inorganic Chemistry Laboratory, National and Kapodistrian University of Athens, Panepistimiopolis, Zografou 15771, Greece.
Inorg Chem. 2022 May 23;61(20):8022-8035. doi: 10.1021/acs.inorgchem.2c00766. Epub 2022 May 12.
Understanding the structure and function of lytic polysaccharide monooxygenases (LPMOs), copper enzymes that degrade recalcitrant polysaccharides, requires the reliable atomistic interpretation of electron paramagnetic resonance (EPR) data on the Cu(II) active site. Among various LPMO families, the chitin-active AA10 shows an intriguing phenomenology with distinct EPR signals, a major rhombic and a minor axial signal. Here, we combine experimental and computational investigations to uncover the structural identity of these signals. X-band EPR spectra recorded at different pH values demonstrate pH-dependent population inversion: the major rhombic signal at pH 6.5 becomes minor at pH 8.5, where the axial signal dominates. This suggests that a protonation change is involved in the interconversion. Precise structural interpretations are pursued with quantum chemical calculations. Given that accurate calculations of Cu -tensors remain challenging for quantum chemistry, we first address this problem via a thorough calibration study. This enables us to define a density functional that achieves accurate and reliable prediction of -tensors, giving confidence in our evaluation of AA10 LPMO models. Large models were considered that include all parts of the protein matrix surrounding the Cu site, along with the characteristic second-sphere features of AA10. The results uniquely identify the rhombic signal with a five-coordinate Cu ion bearing two water molecules in addition to three N-donor ligands. The axial signal is attributed to a four-coordinate Cu ion where only one of the waters remains bound, as hydroxy. Alternatives that involve decoordination of the histidine brace amino group are unlikely based on energetics and spectroscopy. These results provide a reliable spectroscopy-consistent view on the plasticity of the resting state in AA10 LPMO as a foundation for further elucidating structure-property relationships and the formation of catalytically competent species. Our strategy is generally applicable to the study of EPR parameters of mononuclear copper-containing metalloenzymes.
了解溶菌多糖单加氧酶（LPMO）的结构和功能，需要对铜酶活性位点的电子顺磁共振（EPR）数据进行可靠的原子解释。在各种 LPMO 家族中，甲壳素活性的 AA10 表现出一种有趣的现象，具有独特的 EPR 信号，主要是菱形和次要的轴向信号。在这里，我们结合实验和计算研究来揭示这些信号的结构特征。在不同 pH 值下记录的 X 波段 EPR 光谱表明存在 pH 值依赖性的群体反转：在 pH 6.5 时主要的菱形信号在 pH 8.5 时变为次要信号，此时轴向信号占主导地位。这表明质子化变化参与了这种转换。通过量子化学计算进行了精确的结构解释。由于对于量子化学来说，准确计算 Cu -张量仍然具有挑战性，因此我们首先通过彻底的校准研究来解决这个问题。这使我们能够定义一种密度泛函，该密度泛函能够实现 Cu -张量的准确和可靠预测，从而使我们对 AA10 LPMO 模型的评估充满信心。我们考虑了较大的模型，这些模型包括围绕 Cu 位点的蛋白质基质的所有部分，以及 AA10 的特征第二壳层特征。结果唯一地将菱形信号识别为五配位的 Cu 离子，除了三个 N 供体配体外，还带有两个水分子。轴向信号归因于四配位的 Cu 离子，其中只有一个水分子仍然被结合，为羟基。基于能量学和光谱学，不涉及组氨酸支撑氨基酸基团去配位的替代方案不太可能。这些结果为 AA10 LPMO 中静止状态的可塑性提供了可靠的光谱一致视图，为进一步阐明结构-性质关系和形成催化活性物种奠定了基础。我们的策略通常适用于单核含铜金属酶的 EPR 参数研究。
