Structure of the Reduced Copper Active Site in Preprocessed Galactose Oxidase: Ligand Tuning for One-Electron O_2 Activation in Cofactor Biogenesis

摘要

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 O_2-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 le~－ reduction of O_2, and the resulting Cu(Ⅱ)-O_2 (·-) 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 O_2 activation by le~ and the role of the resulting Cu(Ⅱ) in enabling substrate activation for biogenesis.