Formation of a Copper(II)-Tyrosyl Complex at the Active Site of Lytic Polysaccharide Monooxygenases Following Oxidation by H2O2
Formation of a Copper(II)-Tyrosyl Complex at the Active Site of Lytic Polysaccharide Monooxygenases Following Oxidation by H2O2
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DOI：
10.1021/jacs.9b09833
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发表时间：
2019-11-20
影响因子：
15
通讯作者：
Walton, Paul H.
中科院分区：
文献类型：
--
作者：
Paradisi, Alessandro;Johnston, Esther M.;Walton, Paul H.
过氧化氢是含铜裂解多糖单加氧酶(LPMOS)氧化裂解糖基底物的一种共底物。LPMOS与过氧化氢的反应速度很快，但它伴随着酶的快速失活，可能是通过蛋白质氧化。在这里，我们使用UV-Vis，CD，XAS，EPR，VT/VH-MCD和共振拉曼光谱，再加上质谱学和密度泛函计算，证明了在较高的PHS下，AA9 LPMO与H_2O_2反应的产物是单一的铜(II)-酪氨酸自由基物种，对糖基底物的氧化不活跃。铜(II)-酪氨酸基中心需要形成显著的铜(II)-((中心点)OTyr)重叠，这反过来又要求铜(II)的d(x(2)-y(2))自组织元轨道的平面朝向酪氨酸基。我们从Marcus交叉关系中提出，活性中心酪氨酸是由保守的酪氨酸和色氨酸残基组成的“空穴跳跃”电荷转移机制的一部分，该机制可以保护蛋白质活性中心在解偶联周转过程中不被激活。
Hydrogen peroxide is a cosubstrate for the oxidative cleavage of saccharidic substrates by copper-containing lytic polysaccharide monooxygenases (LPMOs). The rate of reaction of LPMOs with hydrogen peroxide is high, but it is accompanied by rapid inactivation of the enzymes, presumably through protein oxidation. Herein, we use UV-vis, CD, XAS, EPR, VT/VH-MCD, and resonance Raman spectroscopies, augmented with mass spectrometry and DFT calculations, to show that the product of reaction of an AA9 LPMO with H2O2 at higher pHs is a singlet Cu(II)-tyrosyl radical species, which is inactive for the oxidation of saccharidic substrates. The Cu(II)-tyrosyl radical center entails the formation of significant Cu(II)-((center dot)OTyr) overlap, which in turn requires that the plane of the d(x(2)-y(2)) SOMO of the Cu(II) is orientated toward the tyrosyl radical. We propose from the Marcus cross-relation that the active site tyrosine is part of a "hole-hopping" charge-transfer mechanism formed of a pathway of conserved tyrosine and tryptophan residues, which can protect the protein active site from inactivation during uncoupled turnover.