Kinetic studies on the redox interconversion of GOase(semi) and GOase(ox) forms of galactose oxidase with inorganic complexes as redox partners

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 NE-PE; 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 similar to 9.5) increases to 4.3 x 10(3) (pH similar to 5.5), while for [Co(phen)(3)](3+) a value of 4.9 x 10(3) (pH similar to 9.5) decreases to 0.04 x 10(3) (pH similar to 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 CH3CO2H and HN3 species bind at the substrate binding site of GOase(semi), thus mimicking the behavior of primary alcohols RCH2OH, the natural substrate of GOase. On coordination, loss of a proton occurs, and inhibition of the oxidation with [Fe(CN)(6)](3-) is observed.