Leiden Institute of Chemistry, Leiden University, Gorlaeus Laboratory, Einsteinweg 55, 2333 CC, Leiden, The Netherlands.
Chemphyschem. 2021 Apr 19;22(8):733-740. doi: 10.1002/cphc.202100063. Epub 2021 Mar 19.
The enzyme laccase catalyzes the reduction of dioxygen to water at the trinuclear copper center (TNC). The TNC comprises a type-3 (T3) and a type-2 (T2) copper site. The paramagnetic NMR spectrum of the small laccase from Streptomyces coelicolor (SLAC) without the substrate shows a mixture of two catalytic states, the resting oxidized (RO) state and the native intermediate (NI) state. An analysis of the resonances of the RO state is reported. In this state, hydrogen resonances only of the T3 copper ligands can be found, in the region of 12-22 ppm. Signals from all six histidine ligands are found and can be attributed to Hδ1, Hβ or backbone amide H nuclei. Two sequence-specific assignments are proposed on the basis of a second-coordination shell variant that also lacks the copper ion at the T1 site, SLAC-T1D/Q291E. This double mutant is found to be exclusively in the RO state, revealing a subtle balance between the RO and the NI states.
漆酶催化三核铜中心（TNC）中二氧的还原为水。TNC 由一个类型 3（T3）和一个类型 2（T2）铜位组成。无底物的深红红螺菌小漆酶（SLAC）的顺磁 NMR 谱显示出两种催化状态的混合物，即静止氧化（RO）状态和天然中间物（NI）状态。报告了对 RO 状态的共振分析。在此状态下，仅可以在 12-22 ppm 的区域找到 T3 铜配体的氢共振。发现所有六个组氨酸配体的信号，可以归因于 Hδ1、Hβ 或骨干酰胺 H 核。基于也缺乏 T1 位铜离子的第二个配位壳变体 SLAC-T1D/Q291E，提出了两个序列特异性分配。发现这种双突变体仅处于 RO 状态，揭示了 RO 和 NI 状态之间的微妙平衡。
