Toggle Main Menu Toggle Search

Open Access padlockePrints

Interconversion of CuI and CuII forms of galactose oxidase: comparison of reduction potentials

Lookup NU author(s): Craig Wright

Downloads

Full text for this publication is not currently held within this repository. Alternative links are provided below where available.


Abstract

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.


Publication metadata

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


Altmetrics

Altmetrics provided by Altmetric


Share