Structure of the Reduced Copper Active Site in Preprocessed Galactose Oxidase: Ligand Tuning for One-Electron O 2 Activation in Cofactor Biogenesis

• Published in: Journal of the American Chemical Society 2016-10, Vol.138 (40), p.13219-13229
• Main Authors: Cowley, Ryan E, Cirera, Jordi, Qayyum, Munzarin F, Rokhsana, Dalia, Hedman, Britt, Hodgson, Keith O, Dooley, David M, Solomon, Edward I
• Format: Article
• Language: English
• Subjects: Catalytic Domain; Coenzymes - biosynthesis; Copper - metabolism; Electron Transport; Galactose Oxidase - chemistry; Galactose Oxidase - metabolism; Ligands; Models, Molecular; Oxygen - metabolism; Quantum Theory
• ISSN: 0002-7863; 1520-5126
• DOI: 10.1021/jacs.6b05792

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 -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 O , and the resulting Cu(II)-O 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 activation by 1e and the role of the resulting Cu(II) in enabling substrate activation for biogenesis.