title: 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 redox-active 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 (lambda 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.
authors.affiliation: Department of Chemistry and Biochemistry, Montana State University, Bozeman, Montana 59717, USA.
authors.forename: Melanie S
authors.initials: MS
authors.lastname: Rogers
authors.forename: Ramón
authors.initials: R
authors.lastname: Hurtado-Guerrero
authors.forename: Susan J
authors.initials: SJ
authors.lastname: Firbank
authors.forename: Malcolm A
authors.initials: MA
authors.lastname: Halcrow
authors.forename: David M
authors.initials: DM
authors.lastname: Dooley
authors.forename: Simon E V
authors.initials: SE
authors.lastname: Phillips
authors.forename: Peter F
authors.initials: PF
authors.lastname: Knowles
authors.forename: Michael J
authors.initials: MJ
authors.lastname: McPherson
mesh_terms: D020134:Catalytic Domain; D003300:Copper / Q000378:metabolism; D003432:Cross-Linking Reagents / Q000378:metabolism; D003545:Cysteine / Q000378:metabolism*; D004578:Electron Spin Resonance Spectroscopy; D005692:Galactose Oxidase / Q000737:chemistry* / Q000378:metabolism*; D007700:Kinetics; D008958:Models, Molecular; D010100:Oxygen / Q000378:metabolism; D011487:Protein Conformation; D013053:Spectrophotometry; D014443:Tyrosine / Q000378:metabolism*
chemical_list: D003432:Cross-Linking Reagents; D014443:Tyrosine; D003300:Copper; D005692:Galactose Oxidase; D003545:Cysteine; D010100:Oxygen
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