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

Abstract

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 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, SLACÀ 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.

Paramagnetic NMR to study iron sulfur proteins: 13C detected experiments illuminate the vicinity of the metal center

“…A complete, multinuclear NMR assignment provides unique site specific information which are essential to study conformational changes and intermolecular interactions. In metalloproteins, the development of experimental approaches to recover the NMR information in the proximity of a paramagnetic center represents a relevant topic, rejuvenated also in the last years by the extensive use of paramagnetic probes to obtain structural information from paramagnetic NMR (Su and Chen 2019 ; Dasgupta et al 2021 ; Parigi et al 2023 ; Ravera et al 2022 ; Clore and Iwahara 2009 ; Clore 2015 ; Ott et al 2021 ; Abelein et al 2022 ; Zambelli et al 2023 ).…”

Structural Insight into the Amino Acid Environment of the Two-Domain Laccase’s Trinuclear Copper Cluster

“…The situation is radically different for 2D laccase. Not only Gln292 (Glu/Asp analog located in the T3 channel) and Asp260 (Asp analog located in the T2 channel near the T2 center), but also Tyr109, Arg240, and His165 side groups (numeration of SgfSL, PDB id 6S0O, Figure 5c) take part in the reduction of oxygen to water [40,42,[64][65][66]. Arg240 (T2 channel, 12Å from the T2 copper ion) and His165 (T3 channel, 4.5Å from the T3β copper ion) are proton donors/acceptors, while Tyr109 (T2 channel, 4Å from the T2 copper ion) can be an alternative electron donor in cases of T1 center functional impairment [40,42,64].…”

“…Arg240 (T2 channel, 12Å from the T2 copper ion) and His165 (T3 channel, 4.5Å from the T3β copper ion) are proton donors/acceptors, while Tyr109 (T2 channel, 4Å from the T2 copper ion) can be an alternative electron donor in cases of T1 center functional impairment [40,42,64]. Ser294 (T3 channel, 6Å from the T3β copper ion) was recently suggested to be involved in proton transfer to TNC SLAC (Ser295 in SgfSL, PDB id 6S0O), but there is no experimental evidence to date [65].…”