Cross-Link Formation of the Cysteine 228−Tyrosine 272 Catalytic Cofactor of Galactose Oxidase Does Not Require Dioxygen
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- 作者：Melanie S. Rogers ; Ramó ; n Hurtado-Guerrero ; Susan J. Firbank ; Malcolm A. Halcrow ; David M. Dooley ; Simon E. V. Phillips ; Peter F. Knowles ; Michael J. McPherson
- 刊名：Biochemistry
- 出版年：2008
- 出版时间：September 30, 2008
- 年：2008
- 卷：47
- 期：39
- 页码：10428-10439
- 全文大小：367K
- 年卷期：v.47,no.39(September 30, 2008)
- ISSN：1520-4995
文摘
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.