Role of the Tyr-Cys cross-link to the active site properties of galactose oxidase.
Role of the Tyr-Cys cross-link to the active site properties of galactose oxidase.
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DOI：
10.1021/ic2022769
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发表时间：
2012-02
影响因子：
4.6
通讯作者：
Dalia Rokhsana;A. Howells;D. Dooley;R. Szilagyi
中科院分区：
文献类型：
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作者：
Dalia Rokhsana;A. Howells;D. Dooley;R. Szilagyi
半乳糖氧化酶 (GO) 活性位点 [Cu(II)-Y·-C] 的催化相关氧化态由反铁磁耦合的 Cu(II) 和翻译后生成的 Tyr-Cys 自由基辅助因子 [Y·-C] 组成。实验表明，Tyr-Cys 交联的硫醚键会影响 GO 活性位点的稳定性、还原电位和催化效率。然而，这些对 GO 活性位点的结构和能量影响的起源尚未得到详细研究。在这里，我们提出了铜和硫 K 边 X 射线吸收数据以及用于评估 GO 中 Tyr-Cys 交联作用的系统计算方法。根据氧化 GO 的硫 K 边 X 射线吸收光谱估计，Tyr-Cys 交联对氧化还原活性轨道的硫贡献约为 24 ± 3%，与 apo-GO (15%) 和 Holo-GO (22%) 计算模型的值相比。 apo-GO 计算模型的结果与先前报告的 apo-GO 值（来自 EPR 的 20 ± 3%）非常一致。令人惊讶的是，Tyr-Cys 交联对全蛋白中 Cu 位点的内球、配位几何形状仅具有最小的影响。它对电子结构的影响更为显着，因为它促进了氧化还原活性轨道离域到源自Cys的硫醚硫上，从而相对于未取代的[Y·]自由基和Cu(II)中心(2210)减少了[Y·-C]自由基和Cu(II)中心(752 cm(-1))之间的自旋耦合。 厘米(-1))。从能量上来说，Tyr-Cys 交联将还原电位降低了约 75 mV（计算值），与没有交联的位点相比，使得全活性位点更容易氧化。总体而言，Tyr-Cys 交联赋予 GO 活性位点独特的基态特性，以非常微妙的方式调整其功能。
The catalytically relevant, oxidized state of the active site [Cu(II)-Y·-C] of galactose oxidase (GO) is composed of antiferromagnetically coupled Cu(II) and a post-translationally generated Tyr-Cys radical cofactor [Y·-C]. The thioether bond of the Tyr-Cys cross-link has been shown experimentally to affect the stability, the reduction potential, and the catalytic efficiency of the GO active site. However, the origin of these structural and energetic effects on the GO active site has not yet been investigated in detail. Here we present copper and sulfur K-edge X-ray absorption data and a systematic computational approach for evaluating the role of the Tyr-Cys cross-link in GO. The sulfur contribution of the Tyr-Cys cross-link to the redox active orbital is estimated from sulfur K-edge X-ray absorption spectra of oxidized GO to be about 24 ± 3%, compared to the values from computational models of apo-GO (15%) and holo-GO (22%). The results for the apo-GO computational models are in good agreement with the previously reported value for apo-GO (20 ± 3% from EPR). Surprisingly, the Tyr-Cys cross-link has only a minimal effect on the inner sphere, coordination geometry of the Cu site in the holo-protein. Its effect on the electronic structure is more striking as it facilitates the delocalization of the redox active orbital onto the thioether sulfur derived from Cys, thereby reducing the spin coupling between the [Y·-C] radical and the Cu(II) center (752 cm(-1)) relative to the unsubstituted [Y·] radical and the Cu(II) center (2210 cm(-1)). Energetically, the Tyr-Cys cross-link lowers the reduction potential by about 75 mV (calculated) allowing a more facile oxidation of the holo active site versus the site without the cross-link. Overall, the Tyr-Cys cross-link confers unique ground state properties on the GO active site that tunes its function in a remarkably nuanced fashion.