Department of Plant and Environmental Sciences, Copenhagen University, Frederiksberg, Denmark.
The Novo Nordisk Foundation Centre for Biosustainability, Technical University of Denmark, Kongens Lyngby, Denmark.
FEBS Lett. 2021 Jun;595(12):1708-1720. doi: 10.1002/1873-3468.14092. Epub 2021 May 14.
The histidine brace (His-brace) is a copper-binding motif that is associated with both oxidative enzymes and proteinaceous copper chaperones. Here, we used biochemical and structural methods to characterize mutants of a His-brace-containing copper chaperone from Pseudomonas fluorescens (PfCopC). A total of 15 amino acid variants in primary and second-sphere residues were produced and characterized in terms of their copper binding and redox properties. PfCopC has a very high affinity for Cu(II) and also binds Cu(I). A high reorganization barrier likely prevents redox cycling and, thus, catalysis. In contrast, mutations in the conserved second-sphere Glu27 enable slow oxidation of ascorbate. The crystal structure of the variant E27A confirmed copper binding at the His-brace. Unexpectedly, Asp83 at the equatorial position was shown to be indispensable for Cu(II) binding in the His-brace of PfCopC. A PfCopC mutant that was designed to mimic the His-brace from lytic polysaccharide monooxygenase-like family X325 did not bind Cu(II), but was still able to bind Cu(I). These results highlight the importance of the proteinaceous environment around the copper His-brace for reactivity and, thus, the difference between enzyme and chaperone.
组氨酸臂（His-brace）是一种与氧化酶和蛋白质铜伴侣都相关的铜结合基序。在这里，我们使用生化和结构方法来研究来自荧光假单胞菌（Pseudomonas fluorescens）的含 His-brace 的铜伴侣（PfCopC）的突变体。在一级和二级结构残基中总共产生了 15 种氨基酸变体，并对其铜结合和氧化还原性质进行了表征。PfCopC 对 Cu(II)具有很高的亲和力，也能结合 Cu(I)。高重组能垒可能阻止了氧化还原循环，从而阻止了催化作用。相比之下，保守的第二配位层残基 Glu27 的突变使抗坏血酸的缓慢氧化成为可能。变体 E27A 的晶体结构证实了在 His-brace 处的铜结合。出乎意料的是，在轴向位置的 Asp83 对于 PfCopC 的 His-brace 中 Cu(II)的结合是不可或缺的。设计成模拟溶菌多糖单加氧酶样家族 X325 的 His-brace 的 PfCopC 突变体不能结合 Cu(II)，但仍然能够结合 Cu(I)。这些结果强调了铜 His-brace 周围蛋白质环境对反应性的重要性，从而解释了酶和伴侣之间的差异。
