The Resting Oxidized State of Small Laccase Analyzed with Paramagnetic NMR Spectroscopy

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 H delta 1, H beta or backbone amide H-N 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.

Automated summary

The enzyme laccase catalyzes the reduction of dioxygen to water at the trinuclear copper center, comprising a type-3 and a type-2 copper site. The paramagnetic NMR spectrum of the small laccase from Streptomyces coelicolor shows a mixture of two catalytic states, with the resting oxidized state exhibiting hydrogen resonances only of the T3 copper ligands. A double mutant variant lacking the copper ion at the T1 site is exclusively in the resting oxidized state, revealing a subtle balance between the resting oxidized and native intermediate states.