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Lookup NU author(s): Dr Susan Firbank
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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 (λmax = 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. © 2008 American Chemical Society.
Author(s): Rogers MS, Hurtado-Guerrero R, Firbank SJ, Halcrow MA, Dooley DM, Phillips SEV, Knowles PF, McPherson MJ
Publication type: Article
Publication status: Published
Journal: Biochemistry
Year: 2008
Volume: 47
Issue: 39
Pages: 10428-10439
ISSN (print): 0006-2960
ISSN (electronic): 1943-295X
Publisher: American Chemical Society
URL: http://dx.doi.org/10.1021/bi8010835
DOI: 10.1021/bi8010835
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