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Lookup NU author(s): Craig Wright, Emeritus Prof Alfred Sykes
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Solutions of galactose oxidase stored in air give in 3-4 h a mix of GOaseox(CuII-Tyr•) and GOasesemi(CuII-Tyr), as a result of processes involving the formation and decay of the CuII-coordinated tyrosyl radical (Tyr•). In this work the two reactions have been studied by UV - vis spectrophotometry and separate rate laws defined. The first involves the "spontaneous" autoreduction of GOaseox to GOasesemi, which in air-free conditions is 100% complete. Rate constants (kred) are dependent on pH, and previously defined acid dissociation constants pK1a = 5.7 (exogenous H2O ligand), and pK2a = 8.0 (axial H+Tyr-495) apply. Values of kred(25 °C) range from 1.55 × 10-4 s-1 (pH 5.5) to 2.69 × 10-4 s-1 (pH 8.6), I = 0.100 M (NaCl). No reaction occurs with N3- or NCS- present in amounts sufficient to give >98% binding at the substrate binding (exogenous) site, while CH3CO2- and phosphate (less extensively bound) also inhibit the reaction. From such inhibition studies K(25 °C) is 161 M-1 at pH 6.4 for acetate (previous value 140 M-1) and 46 M-1 at pH 7.0 for phosphate. No reaction occurs when the disulfide Cys515-Cys518 (10.2 Å from the Cu) is chemically modified with HSPO32-, and electron transfer via the disulfide and exogenous position is proposed (source of the electron not established). The conversion of GOasesemi to GOaseox only occurs with O2 present, when a first-order dependence on [O2] is observed, giving kox(25 °C) = 0.021 M-1 s-1 at pH 7.5. This process is unaffected by NCS- or N3- bound at the exogenous site, and a mechanism involving outer-sphere reaction of O2 to O2- followed by a fast step O2 to H2O2 is proposed. As GOaseox is formed, autoreduction back to GOasesemi occurs, and at pH 7.5 with O2 in large excess (1.13 mM) the maximum conversion to GOaseox is 69%. The kox reaction proceeds to completion with >98% N3- bound at the exogenous site.
Author(s): Wright C, Sykes AG
Publication type: Article
Publication status: Published
Journal: Inorganic Chemistry
Year: 2001
Volume: 40
Issue: 11
Pages: 2528-2533
ISSN (print): 0020-1669
ISSN (electronic): 1520-510X
Publisher: American Chemical Society
URL: http://dx.doi.org/10.1021/ic0011516
DOI: 10.1021/ic0011516
PubMed id: 11350230
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