Structure of the Reduced Copper Active Site in Pre-Processed Galactose Oxidase: Ligand Tuning for One-Electron O2 Activation in Cofactor Biogenesis

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 toward 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 cross-link. 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

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

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

Structure of the Reduced Copper Active Site in Pre-Processed Galactose Oxidase: Ligand Tuning for One-Electron O2 Activation in Cofactor Biogenesis

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 toward 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 cross-link. 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

Structural and Spectroscopic Characterization of Iron(II), Cobalt(II), and Nickel(II) <i>ortho</i>-Dihalophenolate Complexes: Insights into Metal–Halogen Secondary Bonding

Metal complexes incorporating the tris­(3,5-diphenylpyrazolyl)­borate ligand (Tp^Ph2) and <i>ortho</i>-dihalophenolates were synthesized and characterized in order to explore metal–halogen secondary bonding in biorelevant model complexes. The complexes Tp^Ph2ML were synthesized and structurally characterized, where M was Fe­(II), Co­(II), or Ni­(II) and L was either 2,6-dichloro- or 2,6-dibromophenolate. All six complexes exhibited metal–halogen secondary bonds in the solid state, with distances ranging from 2.56 Å for the Tp^Ph2Ni­(2,6-dichlorophenolate) complex to 2.88 Å for the Tp^Ph2Fe­(2,6-dibromophenolate) complex. Variable temperature NMR spectra of the Tp^Ph2Co­(2,6-dichlorophenolate) and Tp^Ph2Ni­(2,6-dichlorophenolate) complexes showed that rotation of the phenolate, which requires loss of the secondary bond, has an activation barrier of ∼30 and ∼37 kJ/mol, respectively. Density functional theory calculations support the presence of a barrier for disruption of the metal–halogen interaction during rotation of the phenolate. On the other hand, calculations using the spectroscopically calibrated angular overlap method suggest essentially no contribution of the halogen to the ligand-field splitting. Overall, these results provide the first quantitative measure of the strength of a metal–halogen secondary bond and demonstrate that it is a weak noncovalent interaction comparable in strength to a hydrogen bond. These results provide insight into the origin of the specificity of the enzyme 2,6-dichlorohydroquinone 1,2-dioxygenase (PcpA), which is specific for <i>ortho</i>-dihalohydroquinone substrates and phenol inhibitors