The trinuclear copper center (TNC) of laccase reduces oxygen to water with very little overpotential. The arrangement of the coppers and ligands in the TNC is known to be from many crystal structures, yet information about possible dynamics of the ligands is absent. Here, we report dynamics at the TNC of small laccase from
Streptomyces coelicolor
using paramagnetic NMR and electron paramagnetic resonance spectroscopy. Fermi contact-shifted resonances tentatively assigned to histidine H
δ
1 display a two-state chemical exchange with exchange rates in the order of 100 s
−1
. In the electron paramagnetic resonance spectra, at least two forms are observed with different
g
z
-values. It is proposed that the exchange processes reflect the rotational motion of histidine imidazole rings that coordinate the coppers in the TNC.
Rapid freeze-quench (RFQ) in combination with electron paramagnetic resonance (EPR) spectroscopy at X-band is a proven technique to trap and characterize paramagnetic intermediates of biochemical reactions. Preparation of suitable samples is still cumbersome, despite many attempts to remedy this problem, and limits the wide applicability of RFQ EPR. We present a method, which improves the collection of freeze-quench particles from isopentane and their packing in an EPR tube. The method is based on sucking the particle suspension into an EPR tube with a filter at the bottom. This procedure results in a significant reduction of the required volume of reactants, which allows the economical use of valuable reactants such as proteins. The approach also enables the successful collection of smaller frozen particles, which are generated at higher flow rates. The method provides for a reproducible, efficient and fast collection of the freeze-quench particles and can be easily adapted to RFQ EPR at higher microwave frequencies than X-band.
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