X-RAY ABSORPTION-EDGE DETERMINATION OF THE OXIDATION-STATE AND COORDINATION-NUMBER OF COPPER - APPLICATION TO THE TYPE-3 SITE IN RHUS-VERNICIFERA LACCASE AND ITS REACTION WITH OXYGEN
X-RAY ABSORPTION-EDGE DETERMINATION OF THE OXIDATION-STATE AND COORDINATION-NUMBER OF COPPER - APPLICATION TO THE TYPE-3 SITE IN RHUS-VERNICIFERA LACCASE AND ITS REACTION WITH OXYGEN
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DOI：
10.1021/ja00255a032
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发表时间：
1987-10-14
影响因子：
15
通讯作者：
SOLOMON, EI
中科院分区：
文献类型：
--
作者：
KAU, LS;SPIRASOLOMON, DJ;SOLOMON, EI
系统研究了19个Cu（I）和40个Cu（II）模型配合物的X射线吸收边特征，并与氧化态和几何构型进行了关联。对具有不同配位数的Cu（I）模型络合物的研究表明，8983-8984-eV峰（指定为Cu 1 s → 4p跃迁）可以在能量、形状和强度上与亚铜离子的连接和位置几何形状相关。这些Cu（I）的边缘功能已定性解释与配位场的概念。 或者，没有Cu（II）络合物表现出低于8985.0eV的峰。对于某些Cu（II）络合物，在8983-8985-eV区域中观察到的有限强度与在λ处的吸收峰的尾部有关。8996 eV，其受赤道配体的共价性影响。这些模型的研究允许一个标准化的差异边缘程序，这是用于定量测定铜（I）含量的混合氧化态组合物中的铜络合物的准确校准。然后使用这种归一化的差异边缘分析来定量地确定2型铜贫化（T2 D）和原生形式的多铜氧化酶，漆树漆酶中的铜位点的氧化态。T2 D漆酶的3型位点被发现被完全还原并且对O2或25倍蛋白当量的铁氰化物的氧化稳定，但它可以通过与过氧化物反应而被氧化。T2 D漆酶的过氧化物滴定导致的330 nm吸收特征的强度的增加被发现与双核铜位点的氧化百分比线性相关。这种相关性表明过氧化物氧化但不结合T3位点。我们已经使用这种相关性来确定天然漆酶，如分离的，含有22 . 3%的减少T3网站和所有的光谱变化后观察到的过氧化物添加到天然漆酶可以解释这些还原位点的氧化。在叠氮化物和过氧化物的存在下，发生进一步还原，并且多达40%的双核铜对稳定在还原状态。这些结果的重要性，以前的报告过氧化物结合在漆酶活性位点进行了讨论。
Cu X-ray absorption edge features of 19 Cu(I) and 40 Cu(II) model complexes have been systemically studied and correlated with oxidation state and geometry. Studies of Cu(I) model complexes with different coordination number reveal that an 8983-8984-eV peak (assigned as the Cu 1s.fwdarw.4p transition) can be correlated in energy, shape, and intensity with ligation and site geometry of the cuprous ion. These Cu(I) edge features have been qualitatively interpreted with ligand field concepts. Alternatively, no Cu(II) complex exhibits a peak below 8985.0eV. The limited intensity observed in the 8983-8985-eV region for some Cu(II) complexes is associated with the tail of an absorption peak at .apprx. 8996 eV which is affected by the covalency of the equatorial ligands. These model studies allow accurate calibration of a normalized difference edge procedure which is used for the quantitative determination of Cu(I) content in copper complexes of mixed oxidation state composition. This normalized difference edge analysis is then used to quantitatively determine the oxidation states of the copper sites in type 2 copper-depleted (T2D) and native forms of the multicopper oxidase, Rhus vernicifera laccase. The type 3 site of the T2D laccase is found to be fully reduced and stable to oxidation by O2 or by 25-fold protein equivalents of ferricyanide, but it can be oxidized by reaction with peroxide. The increase in intensity of the 330-nm absorption feature which results from peroxide titration of T2D laccase is found to correlate linearly with the percent of oxidation of the binuclear copper site. This correlation indicates that peroxide oxidizes but does not bind to the T3 site. We have used this correlation to determine that native laccase, as isolated, contains 22 .+-. 3% reduced T3 sites and that all spectral changes observed upon peroxide addition to native laccase can be accounted for by oxidation of these reduced sites. In the presence of azide and peroxide, further reduction occurs and as much as 40% of the binuclear copper pairs are stabilized in the reduced state. The importance of these results to previous reports of peroxide binding at the laccase active site is discussed.