title: Kinetic Studies on the Redox Interconversion of GOase(semi) and GOase(ox) Forms of Galactose Oxidase with Inorganic Complexes as Redox Partners.
abstract: Redox interconversions between the GOase(semi) (Cu(II), Tyr) and tyrosyl radical containing GOase(ox) (Cu(II), Tyr(*)) oxidation states of the Cu-containing enzyme galactose oxidase (GOase) from Fusarium NRRL 2903 have been studied. The inorganic complexes [Fe(CN)(6)](3)(-) (410 mV), [Co(phen)(3)](3+) (370 mV), [W(CN)(8)](3)(-) (530 mV), and [Co(dipic)(2)](-) (362 mV) (E degrees ' values vs NHE; dipic = 2,6-dicarboxylatopyridine) were used as oxidants for GOase(semi), and [Fe(CN)(6)](4)(-) and [Co(phen)(3)](2+) as reductants for GOase(ox). On oxidation of GOase(semi) a radical is generated at the coordinated phenolate of Tyr-272 to give GOase(ox). The one-electron reduction potential E degrees ' (25 degrees C) for the GOase(ox)/GOase(semi) couple varies with pH and is 400 mV vs NHE at pH 7.5, the smallest value so far observed for a tyrosyl radical. The reactions are very sensitive to pH, or more precisely to pK(a) values of GOase(semi) and GOase(ox), and the charge on the inorganic reagent. For example, with [Fe(CN)(6)](3)(-) as oxidant, the rate constant (25 degrees C)/M(-)(1) s(-)(1) of 0.16 x 10(3) (pH approximately 9.5) increases to 4.3 x 10(3) (pH approximately 5.5), while for [Co(phen)(3)](3+) a value of 4.9 x 10(3) (pH approximately 9.5) decreases to 0.04 x 10(3) (pH approximately 5.5), I = 0.100 M (NaCl). From the kinetics a single GOase(semi) acid dissociation process, pK(a) = 8.0 (average), has been confirmed by UV-vis spectrophotometric studies (7.9). The corresponding value for GOase(ox) is 6.7. No comparable kinetic or spectrophotometric pH dependences are observed with the Tyr495Phe variant, indicating the axial Tyr-495 as the site of protonation. Neutral CH(3)CO(2)H and HN(3) species bind at the substrate binding site of GOase(semi), thus mimicking the behavior of primary alcohols RCH(2)OH, the natural substrate of GOase. On coordination, loss of a proton occurs, and inhibition of the oxidation with [Fe(CN)(6)](3)(-) is observed.
authors.affiliation: Department of Chemistry, The University of Newcastle, Newcastle upon Tyne, NE1 7RU, U.K., and Department of Biochemistry and Molecular Biology, The University of Leeds, Leeds LS2 9JT, U.K.
authors.forename: Colin G.
authors.initials: CG
authors.lastname: Saysell
authors.forename: Christopher D.
authors.initials: CD
authors.lastname: Borman
authors.forename: Andrew J.
authors.initials: AJ
authors.lastname: Baron
authors.forename: Michael J.
authors.initials: MJ
authors.lastname: McPherson
authors.forename: A. Geoffrey
authors.initials: AG
authors.lastname: Sykes