Structure of the Reduced Copper Active Site in Preprocessed Galactose Oxidase: Ligand Tuning for One-Electron O2Activation 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 O2activation by 1e-and the role of the resulting Cu(II) in enabling substrate activation for biogenesis.
Department of Chemistry, Stanford University , Stanford, California 94305, United States.
Ryan E
RE
Cowley
Department of Chemistry, Stanford University , Stanford, California 94305, United States.
Jordi
J
Cirera
Department of Chemistry, Stanford University , Stanford, California 94305, United States.
Munzarin F
MF
Qayyum
Department of Chemistry and Biochemistry, Montana State University , Bozeman, Montana 59717, United States.
Dalia
D
Rokhsana
Department of Chemistry, Stanford University , Stanford, California 94305, United States.|Stanford Synchrotron Radiation Lightsource, SLAC National Accelerator Laboratory, Stanford University , Menlo Park, California 94025, United States.
Britt
B
Hedman
Department of Chemistry, Stanford University , Stanford, California 94305, United States.|Stanford Synchrotron Radiation Lightsource, SLAC National Accelerator Laboratory, Stanford University , Menlo Park, California 94025, United States.
Keith O
KO
Hodgson
Department of Chemistry and Biochemistry, Montana State University , Bozeman, Montana 59717, United States.|University of Rhode Island , Kingston, Rhode Island 02881, United States.
David M
DM
Dooley
Department of Chemistry, Stanford University , Stanford, California 94305, United States.|Stanford Synchrotron Radiation Lightsource, SLAC National Accelerator Laboratory, Stanford University , Menlo Park, California 94025, United States.
Edward I
EI
Solomon
D020134:Catalytic Domain; D003067:Coenzymes / Q000096:biosynthesis*; D003300:Copper / Q000378:metabolism*; D004579:Electron Transport; D005692:Galactose Oxidase / Q000737:chemistry* / Q000378:metabolism*; D008024:Ligands; D008958:Models, Molecular; D010100:Oxygen / Q000378:metabolism*; D011789:Quantum Theory
D003067:Coenzymes; D008024:Ligands; D003300:Copper; D005692:Galactose Oxidase; D010100:Oxygen