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.
Galactose oxidase (GO) is a copper-dependent enzyme that accomplishes 2e− substrate oxidation by pairing a single copper with an unusual cysteinylated tyrosine (Cys-Tyr) redox cofactor. Previous studies have demonstrated that the post-translational biogenesis of Cys-Tyr is copper- and O2-dependent, resulting in a self-processing enzyme system. To investigate the mechanism of cofactor biogenesis in GO, the active-site structure of Cu(I)-loaded GO was determined using X-ray absorption near edge structure (XANES) and extended X-ray absorption fine structure (EXAFS) spectroscopy, and density-functional theory (DFT) calculations were performed on this model. Our results show that the active-site tyrosine lowers the Cu potential to enable the thermodynamically unfavorable 1e− reduction of O2, and the resulting Cu(II)-O2•− is activated towards H-atom abstraction from cysteine. The final step of biogenesis is a concerted reaction involving coordinated Tyr ring deprotonation where Cu(II) coordination enables formation of the Cys-Tyr crosslink. These spectroscopic and computational results highlight the role of the Cu(I) in enabling O2 activation by 1e− and the role of the resulting Cu(II) in enabling substrate activation for biogenesis.
CO dehydrogenase (CODH) is an environmentally crucial bacterial enzyme that oxidizes CO to CO2 at a Mo-Cu active site. Despite the close to atomic resolution structure (1.1 Å), significant uncertainties have remained with regard to the protonation state of the water-derived equatorial ligand coordinated at the Mo-center, as well as the nature of intermediates formed during the catalytic cycle. To address the protonation state of the equatorial ligand, we have developed a realistic in silico QM model (~179 atoms) containing structurally essential residues surrounding the active site. Using our QM model, we examined each plausible combination of redox states (Mo(VI)-Cu(I), Mo(V)-Cu(II), Mo(V)-Cu(I), and Mo(IV)-Cu(I)) and Mo-coordinated equatorial ligands (O(2-), OH(-), H2O), as well as the effects of second-sphere residues surrounding the active site. Herein, we present a refined computational model for the Mo(VI) state in which Glu763 acts as an active site base, leading to a MoO2-like core and a protonated Glu763. Calculated structural and spectroscopic data (hyperfine couplings) are in support of a MoO2-like core in agreement with XRD data. The calculated two-electron reduction potential (E = -467 mV vs. SHE) is in reasonable agreement with the experimental value (E = -558 mV vs. SHE) for the redox couple comprising an equatorial oxo ligand and protonated Glu763 in the Mo(VI)-Cu(I) state and an equatorial water in the Mo(IV)-Cu(I) state. We also suggest a potential role of second-sphere residues (e.g., Glu763, Phe390) based on geometric changes observed upon exclusion of these residues in the most plausible oxidized states.
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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.
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