Cross-Link Formation of the Cysteine 228−Tyrosine 272 Catalytic Cofactor of Galactose Oxidase Does Not Require Dioxygen

Abstract

Galactose oxidase (GO) belongs to a class of proteins that self-catalyze assembly of their redoxactive cofactors from active site amino acids. Generation of enzymatically active GO appears to require at least four sequential post-translational modifications: cleavage of a secretion signal sequence, copper-dependent cleavage of an N-terminal pro sequence, copper-dependent formation of a C228-Y272 thioether bond, and generation of the Y272 radical. The last two processes were investigated using a truncated protein (termed premat-GO) lacking the pro sequence and purified under copper-free conditions. Reactions of premat-GO with Cu(II) were investigated using optical, EPR, and resonance Raman spectroscopy, SDS-PAGE, and X-ray crystallography. Premat-GO reacted anaerobically with excess Cu(II) to efficiently form the thioether bond but not the Y272 radical. A potential C228-copper coordinated intermediate (λ max ) 406 nm) in the processing reaction, which had not yet formed the C228-Y272 cross-link, was identified from the absorption spectrum. A copper-thiolate protein complex, with copper coordinated to C228, H496, and H581, was also observed in a 3 min anaerobic soak by X-ray crystallography, whereas a 24 h soak revealed the C228-Y272 thioether bond. In solution, addition of oxygenated buffer to premat-GO preincubated with excess Cu(II) generated the Y272 radical state. On the basis of these data, a mechanism for the formation of the C228-Y272 bond and tyrosyl radical generation is proposed. The 406 nm complex is demonstrated to be a catalytically competent processing intermediate under anaerobic conditions. We propose a potential mechanism which is in common with aerobic processing by Cu(II) until the step at which the second electron acceptor is required. † This work was supported by the Biotechnology and Biological Sciences Research Council (24/B11662) and NIH (GM 27659). S.J. NIH Public Access Author ManuscriptBiochemistry. Author manuscript; available in PMC 2009 October 15. NIH-PA Author ManuscriptNIH-PA Author Manuscript NIH-PA Author ManuscriptThe range of chemistry available for enzyme catalysis may be extended by post-translational modifications of encoded amino acids, generating new intrinsic cofactor moieties (1). One general class of post-translationally modified cofactor involves protein-derived radicals, including tyrosyl, glycyl, thiyl, and tryptophanyl radicals (2-5). The second class includes proteins that undergo more extensive post-translational modifications derived by covalent modification of an amino acid, or involving peptide cleavage and modification, or new bondforming reactions. Post-translational modification occurs either by a self-processing pathway or via the involvement of accessory enzymes, and both modes of modification may be utilized by an enzyme to facilitate new reactivity and/or provide additional structural functionality.The most frequently modified residue appears to be tyrosine, which is transformed into a variety of novel structures, including cross-links and qui...

Spatially resolved proteomic mapping in living cells with the engineered peroxidase APEX2

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Copper Active Sites in Biology

“…636 The proposed mechanism involves the deprotonation of the OH group of the sugar by an axial tyrosine (Y495) upon coordination to the Cu(II), a stereospecific pro-S hydrogen atom abstraction by the Cys-Tyr radical cofactor, an inner sphere electron transfer by the substrate radical reducing Cu(II) to Cu(I) and finally the dissociation of the aldehyde product (Figure 105). 622 …”

“…636 To study the possible role of Cu(II) in biogenesis, both aerobic and anaerobic loading experiments have been done with preprocessed GO and Cu(II).…”

“…After 24 h, the anaerobic Cu(II) soaked crystals show that the Cu-S bond is broken, the thioether crosslink is formed, and Tyr272 is coordinated to the Cu(II) in the equatorial plane (Figure 110C). 636 …”

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