Structure of the oxidized active site of galactose oxidase from realistic in silico models.

A systematic in silico approach is employed to generate an accurate model for the catalytically important oxidized state of galactose oxidase (GO) using spectroscopically calibrated hybrid density-functional theory. GO displays three distinct oxidation states: oxidized [Cu(II)-Y*], semireduced [Cu(II)-Y], and fully reduced [Cu(I)-Y], but only the [Cu(II)-Y*] and the [Cu(I)-Y] states are assumed to be involved in catalysis. We have developed multiple models for the oxidized [Cu(II)-Y*] state, whose structure has not yet been fully characterized. These models were evaluated by comparison of calculated and experimental structural data, singlet-triplet energy gaps, and electronic transitions for the antiferromagnetically coupled oxidized [Cu(II)-Y*] state. An extended model system that includes explicit solvent molecules and second coordination sphere residues (R330, Y405, and W290) is essential to obtain the correct electronic structure of the active site. The model with all the residues that have been shown to affect the radical stability and catalysis resulted in a singlet ground state with the radical centered on the Y272-C228 cofactor. The optimized structure of the oxidized GO [Cu(II)-Y*] reveals a five-coordinated square pyramidal coordination geometry very similar to [Cu(II)-Y] with considerably different Cu-ligand distances. The hydrogen-bonding interactions involving Y495 modulates the spin density distribution and the singlet-triplet energy gaps. The final model as the most reasonable structure of the oxidized [Cu(II)-Y*] state in GO reproduces the spectroscopic signature of oxidized GO.