Instituto de Tecnologia Química e Biológica, Universidade Nova de Lisboa, Av. da República, 2780-157 Oeiras, Portugal.
Biochem Biophys Res Commun. 2012 Jun 8;422(3):442-6. doi: 10.1016/j.bbrc.2012.05.011. Epub 2012 May 8.
Copper is a redox-active metal and the main player in electron transfer reactions occurring in multicopper oxidases. The role of copper in the unfolding pathway and refolding of the multicopper oxidase CotA laccase in vitro was solved using double-jump stopped-flow experiments. Unfolding of apo- and holo-CotA was described as a three-state process with accumulation of an intermediate in between the native and unfolded state. Copper stabilizes the native holo-CotA but also the intermediate state showing that copper is still bound to this state. Also, copper binds to unfolded holo-CotA in a non-native coordination promoting CotA aggregation and preventing refolding to the native structure. These results gather information on unfolding/folding pathways of multicopper oxidases and show that copper incorporation in vivo should be a tight controlled process as copper binding to the unfolded state under native conditions promotes protein aggregation.
铜是一种氧化还原活性金属，也是多铜氧化酶中电子转移反应的主要参与者。使用双跳跃停流实验解决了铜在体外多铜氧化酶 CotA 漆酶展开途径和重折叠中的作用。去辅基和全辅基 CotA 的展开被描述为一个三态过程，在天然状态和展开状态之间积累一个中间状态。铜稳定天然全辅基 CotA，但也稳定中间状态，表明铜仍然与该状态结合。此外，铜以非天然配位结合展开的全辅基 CotA，促进 CotA 聚集，并阻止其重折叠为天然结构。这些结果收集了多铜氧化酶展开/折叠途径的信息，并表明体内铜的掺入应该是一个严格控制的过程，因为在天然条件下铜与展开状态结合会促进蛋白质聚集。
