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Autoredox interconversion of two galactose oxidase forms GOaseox and GOasesemi with and without dioxygen

Lookup NU author(s): Craig Wright, Emeritus Prof Alfred Sykes

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Abstract

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.


Publication metadata

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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