Structure of the Oxidized Active Site of Galactose Oxidase from Realistic In Silico Models

Abstract

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.

Sulfanyl stabilization of copper-bonded phenoxyls in model complexes and galactose oxidase

“…The nature of the IVCT transition in ½1 SR2 þ lends credence to the contributions of similar transitions from the tyrosinate to the cysteine-modified tyrosyl radical in GO oxy , a pointed disputed in the literature (7,42). The coupling between the phenoxyl radical and the Cu II unpaired spins in oxidized GO precludes its characterization by EPR spectroscopy.…”

“…The electronic spectrum of the fully oxidized GO exhibits multiple overlapping features in the vis-NIR region (8;000-20;000 cm −1 , 3;200 M −1 cm −1 ) (1) that are attributed alternatively to either sulfur-aryl π → π Ã (7,8) or phenolate-phenoxyl IVCT (42) transitions. The electronic spectra of ½1 SR2 þ exhibit aryl π → π Ã transitions [ Fig.…”

“…660-1,000 mV) (1, 6). Delocalization of the tyrosyl radical onto the thioether bridge, which is predicted by density functional theory (DFT) computations (7,8) and EPR studies of copper-free GO (9), is postulated to contribute to the energetic stabilization of GO oxy . This postulate is consistent with the in-plane conformation of the cysteine-tyrosine cross-link observed in all known crystal structures of GO (3-5), yet the extent of hole delocalization on sulfur and consequent energetic influence in copper-bonded phenoxyls including GO oxy remain unknown (9, 10).…”

Metal Coordinated Phenoxyl Radicals

“…Strong antiferromagnetic exchange coupling between the tyrosyl radical and the paramagnetic copper(II) ion induces a diamagnetic ground state, with a singlet-triplet splitting greater than 200 cm −1 . 51,52 Catalysis of galactose oxidase proceeds in a ping-pong turnover reaction (Figure 8.5). 53 -55 In the first half-reaction the alcohol binds to the active site and the tyrosyl radical abstracts a hydrogen atom from the substrate (the thermodynamic affinity of a tyrosyl radical for a hydrogen atom is high).…”

Gas Phase Investigation of [(Cu2+,Ni2+-Gly-Gly-His)-3H+]-1Complex by Electrospray Ionization MS/MS and MS/MS/MS

“…12,13 Theoretical studies concerning metal-oligopeptide structure and metal-ligand coordination geometry have also been performed through molecular dynamics simulations and ab initio calculations. [14][15][16][17] Structures, molecular orbital and stabilization energies of metal-oligopeptides are reported by the research groups.…”