Interconversion of Cu(I) and Cu(II) forms of galactose oxidase: comparison of reduction potentials.
A procedure for the preparation of the fully reduced Cu(I) form of galactose oxidase, GOase(red), involving reduction of GOase(semi) (or GOase(ox)) with non-coordinating [Ru(NH(3))(6)](2+) (51 mV vs. nhe) is described. Air-free conditions and a two-fold excess of [Ru(NH(3))(6)](2+) give a stable product with no further UV-Vis changes over >1.5 h. Rate constants for the reduction of GOase(semi) (k(f)=860 M(-1) s(-1)) give a first-order [H(+)]-dependence (pK(1a)=7.9), but the reverse process involving [Ru(NH(3))(6)](3+) oxidation of GOase(red) (k(b)=18.6 M(-1) s(-1)) is independent of pH (5.5 to 9.5). The reduction potential E(2)(o)' (vs. nhe) for the GOase(semi)/GOase(red) (i.e. Cu(II)/Cu(I)) couple is 149 mV at pH 7.5, which varies from 160 mV (pH 5.5) to 120 mV (pH 10.5), suggesting pK(1a) (GOase(semi)) and pK(2a) (GOase(red)) acid dissociation constants both involving Tyr-495. It is concluded that pK(2a) is for acid dissociation of uncoordinated H(+)Tyr-495. Consistent with this interpretation rate constants/M(-1) s(-1) for the GOase(semi) Tyr495 Phe variant, k(f)=1.59x10(3) and k(b)=16.1, respectively, are independent of pH and give a reduction potential of 169 mV. Comparisons are made of reduction potentials (E(1)(o)'/mV pH 7.5) for the GOase(ox)/GOase(semi) (i.e. Tyr(.)/Tyr) couple, and are for the Cys228Gly variant (630), for enzyme with N(3)(-) for H(2)O 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 E(1)(o)' of 400 mV for the Tyr(.)/Tyr couple. At pH 7.5 the reduction potential for the two-equivalent GOase(ox)/GOase(red) couple is calculated to be 275 mV. The rate constant for the reaction of GOase(red) with GOase(ox) is 4.4x10(3) M(-1) s(-1) at pH 7.5.
Wright
C
C
Department of Chemistry, The University of Newcastle, Newcastle upon Tyne NE1 7RU, UK.
Sykes
A G
AG
D003300:Copper / Q000737:chemistry*; D005692:Galactose Oxidase / Q000737:chemistry* / Q000302:isolation & purification; D007700:Kinetics; D010084:Oxidation-Reduction
D003300:Copper; D005692:Galactose Oxidase