Structure of the Reduced Copper Active Site in Preprocessed Galactose Oxidase: Ligand Tuning for One-Electron O2 Activation in Cofactor Biogenesis
Ryan E. Cowley,† Jordi Cirera,†,⊥ Munzarin F. Qayyum,† Dalia Rokhsana,‡,# Britt Hedman,†,§
Keith O. Hodgson,†,§ David M. Dooley,*,‡,∥ and Edward I. Solomon*,†,§
†Department of Chemistry, Stanford University, Stanford, California 94305, United States
‡Department of Chemistry and Biochemistry, Montana State University, Bozeman, Montana 59717, United States
§Stanford Synchrotron Radiation Lightsource, SLAC National Accelerator Laboratory, Stanford University, Menlo Park, California
94025, United States
∥University of Rhode Island, Kingston, Rhode Island 02881, United States
ABSTRACT
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 posttranslational 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.
DOI: 10.1021/jacs.6b05792
Ryan E. Cowley,† Jordi Cirera,†,⊥ Munzarin F. Qayyum,† Dalia Rokhsana,‡,# Britt Hedman,†,§
Keith O. Hodgson,†,§ David M. Dooley,*,‡,∥ and Edward I. Solomon*,†,§
†Department of Chemistry, Stanford University, Stanford, California 94305, United States
‡Department of Chemistry and Biochemistry, Montana State University, Bozeman, Montana 59717, United States
§Stanford Synchrotron Radiation Lightsource, SLAC National Accelerator Laboratory, Stanford University, Menlo Park, California
94025, United States
∥University of Rhode Island, Kingston, Rhode Island 02881, United States
ABSTRACT
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 posttranslational 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.
DOI: 10.1021/jacs.6b05792
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