Structural and mechanistic insights into the oxy form of tyrosinase from molecular dynamics simulations

The first, long time scale (16-ns) ligand field molecular dynamics (LFMD) simulations of the oxy form of tyrosinase are reported. The calculations use our existing type 3 copper force field for the peroxido-bridged [Cu2O2]2+ unit which is here translated from MMFF into the AMBER format together with a new charge scheme. The protein secondary and tertiary structures are not significantly altered by removing the ‘caddie’ protein, ORF378, which must be bound to tyrosinase before crystals will grow. A comprehensive principal component analysis of the Cartesian coordinates from the final 8 ns shows that the protein backbone is relatively rigid. However, the significant butterfly fold of the [Cu2O2]2+ moiety observed in the X-ray structure, presumably due to the caddie protein tyrosine at the active site, is absent in the simulations. LFMD gives a clear and persistent distinction between equatorial and axial Cu–N distances, with the latter about 0.2 Å longer and remaining syn to each other. However, the two coordination spheres display important differences. LFMD simulations of the symmetric model complex [μ-η2:μ2-O2{Cu(Meim)3}2]2+ (Meim is 5-methyl-1H-imidazole) provide a mechanism for syn–anti interchange of axial ligands which suggests, in combination with the old experimental X-ray data, the new LFMD simulations and traditional coordination chemistry arguments, that His54 on CuA is ‘insipiently axial’ and that a combination of a butterfly distortion of the [Cu2O2]2+ group and a rotation of the CuA(His)3 moiety converts the vacant, initially axial, binding site on CuA into a much more favourable equatorial site.