Paramagnetic Properties of the Halide-Bound Derivatives of Oxidised Tyrosinase Investigated by 1H NMR Spectroscopy

The 1H NMR relaxation characteristics of the histidines in the oxidised type‐3 copper site of tyrosinase (Tymet) from the bacterium Streptomyces antibioticus in the halide‐bound forms (TymetX with X = F−, Cl−, Br−) have been determined and analysed. The 1H NMR spectra of the TymetX species display remarkably sharp, well‐resolved, paramagnetically shifted 1H signals, which originate from the protons of the six His residues coordinated to the two CuII ions in the type‐3 centre. From the temperature‐dependence of the 1H paramagnetic shifts the following values for the exchange‐coupling parameter −2J were determined: 260 (TymetF), 200 (TymetCl) and 162 cm−1 (TymetBr). The 1H T1 relaxation is dipolar in origin and correlates with the CuH distances. Electronic relaxation times τS derived from the 1H T1 data amount to about 10−11 s and follow the order TymetF>TymetCl>TymetBr. They are two orders of magnitude shorter than the τS values reported for mononuclear copper systems, in accordance with the sharpness of the 1H signals. The results corroborate the Cu2 bridging mode of the halide ions. On the basis of the measured hyperfine interaction constants for the ligand histidine nuclei, it is concluded that 70–80 % of the spin density in the excited triplet state resides on the two copper ions and the bridging atoms. A couple relaxes faster: Paramagnetic 1H NMR spectroscopy of copper enzymes is often prohibited by excessive line broadening. Spin exchange in the Cu2 centre of tyrosinase, however, is responsible for fast electron‐spin relaxation and a concomitant sharpening of the NMR spectra. Relaxation characteristics, spin‐density distribution, and exchange coupling have been determined for the halide‐bridged (F−, Cl−, Br−) Cu2 centre in tyrosinase (see picture). Roughly 80 % of the spin density resides on the metals.