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Lookup NU author(s): Craig Wright
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A procedure for the preparation of the fully reduced CuI form of galactose oxidase, GOasered, involving reduction of GOasesemi (or GOaseox) with non-coordinating [Ru(NH3)6]2+ (51 mV vs. nhe) is described. Air-free conditions and a two-fold excess of [Ru(NH3)6]2+ give a stable product with no further UV-Vis changes over >1.5 h. Rate constants for the reduction of GOasesemi (kf=860 M-1 s-1) give a first-order [H+]-dependence (pK1a=7.9), but the reverse process involving [Ru(NH3)6]3+ oxidation of GOasered (kb=18.6 M-1 s-1) is independent of pH (5.5 to 9.5). The reduction potential E2o′ (vs. nhe) for the GOasesemi/GOasered (i.e. CuII/CuI) couple is 149 mV at pH 7.5, which varies from 160 mV (pH 5.5) to 120 mV (pH 10.5), suggesting pK1a (GOasesemi) and pK2a (GOasered) acid dissociation constants both involving Tyr-495. It is concluded that pK2a is for acid dissociation of uncoordinated H+Tyr-495. Consistent with this interpretation rate constants/M-1 s-1 for the GOasesemi Tyr495 Phe variant, kf=1.59×103 and kb=16.1, respectively, are independent of pH and give a reduction potential of 169 mV. Comparisons are made of reduction potentials (E1o′/mV pH 7.5) for the GOaseox/GOasesemi (i.e. Tyr·/Tyr) couple, and are for the Cys228Gly variant (630), for enzyme with N3- for H2O at the substrate binding exogenous site (393), and for apo-protein (570). These compare with previously reported values for the variants Trp290His (730) and Tyr495Phe (450), and together serve to quantify different contributions to the unusually small E1o′ of 400 mV for the Tyr·/Tyr couple. At pH 7.5 the reduction potential for the two-equivalent GOaseox/GOasered couple is calculated to be 275 mV. The rate constant for the reaction of GOasered with GOaseox is 4.4×103 M-1 s-1 at pH 7.5. Copyright © 2001 Elsevier Science B.V.
Author(s): Wright C, Sykes AG
Publication type: Article
Publication status: Published
Journal: Journal of Inorganic Biochemistry
Year: 2001
Volume: 85
Issue: 4
Pages: 237-243
Print publication date: 01/01/2001
ISSN (print): 0162-0134
ISSN (electronic): 1873-3344
Publisher: Elsevier
URL: http://dx.doi.org/10.1016/S0162-0134(01)00214-8
DOI: 10.1016/S0162-0134(01)00214-8
PubMed id: 11551381
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