Clark Kevin M, Yu Yang, van der Donk Wilfred A, Blackburn Ninian, Lu Yi
University of Illinois-Urbana, Department of Biochemistry, Urbana, IL61801; USA.
University of Illinois-Urbana. Center for Biophysics and Computational Biology, Urbana, IL 61801, USA.
Inorg Chem Front. 2014 Feb 1;1(2):153-158. doi: 10.1039/C3QI00096F.
The Cu-S interaction is known to play a dominant role in defining the type 1 (T1) blue copper center with respect to both its electronic structure and electron transfer function. Despite this importance, its role has yet to be probed by mutagenesis studies without dramatic change of its T1 copper character. We herein report replacement of the conserved Cys112 in azurin with the nonproteinogenic amino acid homocysteine. Based on electronic absorption, electron paramagnetic resonance, and extended x-ray absorption fine structural spectroscopic studies, this variant displays typical type 1 copper site features. Surprisingly, instead of increasing the strength of the Cu-sulfur interaction by the introduction of the extra methylene group, the Cys112Hcy azurin showed a decrease in the covalent interaction between S and Cu(II) when compared with the WT S-Cu(II) interaction. This is likely due to geometric adjustment of the center that resulted in the copper ion moving out of the trigonal plane defined by two histidines and one Hcy and closer to Met121. These structural changes resulted in an increase of reduction potential by 35 mV, consistent with lower Cu-S covalency. These results suggest that the Cu-S interaction is close to being optimal in native blue copper protein. It also demonstrates the power of using nonproteinogenic amino acids in addressing important issues in bioinorganic chemistry.
已知铜 - 硫相互作用在决定1型（T1）蓝铜中心的电子结构和电子转移功能方面起着主导作用。尽管其具有重要性，但在不显著改变其T1铜特性的情况下，其作用尚未通过诱变研究进行探究。我们在此报告了将天青蛋白中保守的半胱氨酸112替换为非蛋白质氨基酸高半胱氨酸。基于电子吸收、电子顺磁共振和扩展X射线吸收精细结构光谱研究，该变体显示出典型的1型铜位点特征。令人惊讶的是，与野生型硫 - 铜（II）相互作用相比，半胱氨酸112高半胱氨酸天青蛋白并没有通过引入额外的亚甲基来增强铜 - 硫相互作用，反而显示出硫与铜（II）之间的共价相互作用减弱。这可能是由于中心的几何调整导致铜离子移出由两个组氨酸和一个高半胱氨酸定义的三角平面，并更靠近甲硫氨酸121。这些结构变化导致还原电位增加了35 mV，这与较低的铜 - 硫共价性一致。这些结果表明，在天然蓝铜蛋白中铜 - 硫相互作用接近最佳状态。它还展示了使用非蛋白质氨基酸解决生物无机化学中重要问题的能力。
