Solutions of galactose oxidase stored in air give in 3-4 h a mix of GOase(ox)(Cu-II-Tyr(.)) and GOase(semi)(Cu-II-Tyr), as a result of processes involving the formation and decay of the Cu-II-coordinated tyrosyl radical (Tyr(.)). In this work the two reactions have been studied by UV-vis spectrophotometry and separate rate laws defined. The first involves the "spontaneous" autoreduction of GOase(ox) to GOase(semi), which in air-free conditions is 100% complete. Rate constants (k(red)) are dependent on pH, and previously defined acid dissociation constants pKi(1a) = 5.7 (exogenous H2O ligand), and pK(2a) = 8.0 (axial. H(+)Tyr-495) apply. Values of k(red)(25 degreesC) range from 1.55 x 10(-4) s(-1) (pH 5.5) to 2.69 x 10(-4) s(-1) (pH 8.6), I = 0.100 M (NaCl). No reaction occurs with N-3(-) or NCS- present in amounts sufficient to give > 98% binding at the substrate binding (exogenous) site, while CH3CO2- and phosphate (less extensively bound) also inhibit the reaction. From such inhibition studies K(25 degreesC) is 161 M-1 at pH 6.4 for acetate (previous value 140 M-1) and 46 M-1 at pH 7.0 for phosphate. No reaction occurs when the disulfide Cys515-Cys518 (10.2 Angstrom from the Cu) is chemically modified with HSPO32-, and electron transfer via the disulfide and exogenous position is proposed (source of the electron not established). The conversion of GOase(semi) to GOase(ox) only occurs with O-2 present, when a first-order dependence on [O-2] is observed, giving k(ox)(25 degreesC) = 0.021 M-1 s(-1) at pH 7.5. This process is unaffected by NCS-or N-3(-) bound at the exogenous site, and a mechanism involving outer-sphere reaction of O-2 to O-2(-) followed by a fast step O-2 to H2O2 is proposed. As GOase(ox) is formed, autoreduction back to GOase(semi) occurs, and at pH 7.5 with O-2 in large excess (1.13 mM) the maximum conversion to GOase(ox) is 69%. The k(ox) reaction proceeds to completion with >98% N-3(-) bound at the exogenous site.