Abstract. Laccases efficiently reduce dioxygen to water in an active site containing a tri-nuclear copper centre (TNC). The dynamics of the protein matrix is a
determining factor in the efficiency in catalysis. To probe mobility, nuclear magnetic resonance (NMR) spectroscopy is highly suitable. However, several factors complicate the assignment of resonances to active site nuclei in laccases. The paramagnetic nature causes large shifts and line broadening. Furthermore, the presence of
slow chemical exchange processes of the imidazole rings of copper ligand
results in peak doubling. A third complicating factor is that the enzyme occurs in two states, the native intermediate (NI) and resting oxidized (RO)
states, with different paramagnetic properties. The present study aims at
resolving the complex paramagnetic NMR spectra of the TNC of Streptomyces coelicolor small laccase
(SLAC). With a combination of paramagnetically tailored NMR experiments, all
eight His Nδ1 and Hδ1 resonances for the NI state are
identified, as well as His Hβ protons for the RO state. With the help
of second-shell mutagenesis, selective resonances are tentatively assigned to the histidine ligands of the copper in the type-2 site. This study
demonstrates the utility of the approaches used for the sequence-specific assignment of the paramagnetic NMR spectra of ligands in the TNC that
ultimately may lead to a description of the underlying motion.
Solution-State Proton Nuclear Magnetic Resonance (NMR) Spectroscopic Studies of the Active Site of Myoglobins in Various Ligated States: Models for Macromolecule-Substrate Binding and Advancement of Paramagnetic NMR Techniques
