Role of the Tyr-Cys Cross-link to the Active Site Properties of Galactose Oxidase

Abstract

The catalytically relevant, oxidized state of the active site [Cu(II)-Y·-C] of galactose oxidase (GO) is composed of antiferromagnetically coupled Cu(II) and a post-translationally generated Tyr-Cys radical cofactor [Y·-C]. The thioether bond of the Tyr-Cys cross-link has been shown experimentally to affect the stability, the reduction potential, and the catalytic efficiency of the GO active site. However, the origin of these structural and energetic effects on the GO active site has not yet been investigated in detail. Here we present copper and sulfur K-edge X-ray absorption data and a systematic computational approach for evaluating the role of the Tyr-Cys cross-link in GO. The sulfur contribution of the Tyr-Cys cross-link to the redox active orbital is estimated from sulfur K-edge X-ray absorption spectra of oxidized GO to be about 24 ± 3%, compared to the values from computational models of apo-GO (15%) and holo-GO (22%). The results for the apo-GO computational models are in good agreement with the previously reported value for apo-GO (20 ± 3% from EPR). Surprisingly, the Tyr-Cys cross-link has only a minimal effect on the inner sphere, coordination geometry of the Cu site in the holo-protein. Its effect on the electronic structure is more striking as it facilitates the delocalization of the redox active orbital onto the thioether sulfur derived from Cys, thereby reducing the spin coupling between the [Y·-C] radical and the Cu(II) center (752 cm(-1)) relative to the unsubstituted [Y·] radical and the Cu(II) center (2210 cm(-1)). Energetically, the Tyr-Cys cross-link lowers the reduction potential by about 75 mV (calculated) allowing a more facile oxidation of the holo active site versus the site without the cross-link. Overall, the Tyr-Cys cross-link confers unique ground state properties on the GO active site that tunes its function in a remarkably nuanced fashion.

N3S-ligated Copper(II) Complex Catalyzed Selective Oxidation of Benzylic Alcohols to Aldehydes under Mild Reaction Conditions

“…Very recently, Rokhsana et al have reported that the Tyr-Cys cross linked sulfur environment is essential for fine-tuning the electronic structure of the GO's active site, 16 and the sulfur environment may be responsible for the selective oxidation of benzylic alcohols over the aliphatic alcohols. We hypothesize that a Cu complex with an N3S coordination environment may mimic the activity of the active site of GO, leading to similar catalytic activity.…”

Combined Mössbauer spectroscopic, multi-edge X-ray absorption spectroscopic, and density functional theoretical study of the radical SAM enzyme spore photoproduct lyase

“…The electrostatic environment of the protein matrix was approximated using a water-based polarizable continuum model [56–58] with a commonly used dielectric constant of ε = 10 for polar protein environment [59]. The effect of the specific value of the dielectric constant in going from 10 to 40 units for the relative energies is expected to be at most 0.2 eV or 20 kJ/mol [60]. …”

Recent advances in phenoxyl radical complexes of salen-type ligands as mixed-valent galactose oxidase models

“…DFT calculations suggest that an orientation of the less sterically demanding -SMe substituent in plane with the phenoxyl ring maximizes S p -orbital overlap with the aromatic π system in [Cu 12 SMe2 ] + , allowing for greater delocalization of the hole onto the sulfur atoms [19]. In a recent sulfur K-edge XAS study of GO ox itself, Rokhsana et al report a sulfur contribution from the Cys-Tyr cross link to the tyrosyl hole of 24 ± 3 % [50]. This value is in good agreement with previous electron paramagnetic resonance (EPR) studies of apo-GO ox that predicted a value of 20 ± 3 % [51].…”