Center for Interdisciplinary Biosciences, Technology and Innovation Park P.J. Šafárik University, Jesenna 5, 041 54 Košice, Slovakia.
Department of Biology and Biological Engineering, Division of Chemical Biology, Chalmers University of Technology, 412 96 Gothenburg, Sweden.
Int J Mol Sci. 2018 Jan 16;19(1):269. doi: 10.3390/ijms19010269.
Fet3p is a multicopper oxidase that contains three cupredoxin-like domains and four copper ions located in three distinct metal sites (T1 in domain 3; T2 and the binuclear T3 at the interface between domains 1 and 3). To probe the role of the copper sites in Fet3p thermodynamic stability, we performed urea-induced unfolding experiments with holo-, apo- and three partially-metallated (T1, T2 and T1/T2 sites depleted of copper) forms of Fet3p. Using a combination of spectroscopic probes (circular dichroism, fluorescence intensity and maximum, 8-anilinonaphthalene-1-sulfonic acid (ANS) emission, oxidase activity and blue color), we reveal that all forms of Fet3p unfold in a four-state reaction with two partially-folded intermediates. Using phase diagrams, it emerged that Fet3p with all copper sites filled had a significantly higher stability as compared to the combined contributions of the individual copper sites. Hence, there is long-range inter-domain communication between distal copper sites that contribute to overall Fet3p stability.
Fet3p 是一种多铜氧化酶，包含三个铜蓝蛋白样结构域和四个位于三个不同金属位点的铜离子（T1 在结构域 3 中；T2 和双核 T3 在结构域 1 和 3 之间的界面处）。为了研究铜位点在 Fet3p 热力学稳定性中的作用，我们对全酶、脱辅基酶和三种部分金属化（T1、T2 和 T1/T2 位点耗尽铜）形式的 Fet3p 进行了尿素诱导的展开实验。使用光谱探针（圆二色性、荧光强度和最大值、8-苯胺基萘-1-磺酸（ANS）发射、氧化酶活性和蓝色）的组合，我们揭示了所有形式的 Fet3p 都以四级反应展开，其中有两个部分折叠的中间体。通过相图，结果表明，与单个铜位点的贡献相比，所有铜位点都填满的 Fet3p 具有更高的稳定性。因此，远端铜位点之间存在长程的结构域间通讯，这有助于整体 Fet3p 的稳定性。
