Cowley, Ryan E.Cirera Fernández, JordiQayyum, Munzarin F.Rokhsana, DaliaHedman, BrittHodgson, Keith O.Dooley, David M.Solomon, Edward I.2019-02-042019-02-042016-09-140002-7863https://hdl.handle.net/2445/127866Galactose 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.11 p.application/pdfeng(c) American Chemical Society , 2016EnzimsTransport d'electronsCatàlisiEnzymesElectron transportCatalysisinfo:eu-repo/semantics/article6673162019-02-04info:eu-repo/semantics/openAccess27626829