Paramagnetic NMR Spectroscopy of a Tri-Nuclear Copper Center from a Multicopper Oxidase, Laccase

Abstract

Among metalloproteins multicopper oxidases and especially laccases are noteworthy because of their wide application in the fields of green chemistry, medicine, bioremediation and enzymatic biofuel cells. Laccases have a type 1 (T1) copper site for oxidation of substrates and a tri-nuclear copper center (TNC) for oxygen reduction reaction. The TNC comprises a binuclear type 3 (T3) site and a mononuclear type 2 (T2) site. We report the paramagnetic NMR spectra of a laccase from Streptomyces coelicolor, in which the Fermi-contact shifted (FCS) resonances were assigned to the coordinating histidine Nδ1/Hδ1 nuclei using tailored 1H-1H EXSY and 1H-15N HMQC experiments. The pNMR spectra of the resting oxidized state and the native intermediate state are shown. The FCS resonances from the NI state display two-state chemical exchange with exchange rates in the order of 100 s−1. This is supported by the observation of two different gz values from the EPR spectra. It is proposed that the exchange processes reflect rotational motion of histidine imidazole rings that coordinate the coppers in the TNC. To our knowledge this is the first report that shows multidimensional paramagnetic NMR spectra of the tri-nuclear copper center and the presence of chemical exchange in the tri-nuclear copper centre of a laccase. The potential catalytic relevance is under study.