Thermodynamic and kinetic studies on the X- = NCS-, N-3(-), and CH3CO2- replacement of H2O/OH- at the Cu-II exogenous site of the tyrosyl-radical-containing enzyme galactose oxidase (GOase(ox)) from Fusarium (NRR 2903), have been studied by methods involving UV-vis spectrophotometry (25 degreesC), pH range 5.5-8.7, I = 0.100 M (NaCl). In the case of N-3(-) and CH2CO2- previous X-ray structures have confirmed coordination at the exogenous H2O/OH- site. From the effect of pH on the UV-vis spectrum of GOase(ox) under buffer-free conditions, acid dissociation constants of 5.7 (pK(1a); coordinated H2O) and 7.0 (pK(2a); H(+)Tyr-495) have been determined. At pH 7.0 formation constants K(25 degreesC)/M-1 are NCS- (480), N-3(-) (1.98 x 10(4)), and CH3CO2- (104), and from the variations in K with pH the same two pK(a) values are seen to apply. No pK(1a) is observed when X- is coordinated. From equilibration stopped-flow Studies rate constants at pH 7.0 for the formation reaction k(f)(25 degreesC)/M-1 s(-1) are NCS- (1.13 x 10(4)) and N-3(-) (5.2 x 10(5)). Both K and k(f) decrease with increasing pH, consistent with the electrostatic effect of replacing H2O by OH-. In the case of the GOase(ox) Tyr495Phe variant pK(1a) is again 5.7, but no pK(2a) is observed, confirming the latter as acid dissociation of protonated Tyr-395. At pH 7.0, K for the reaction of four-coordinate GOase(ox) Tyr495Phe with NCS- (1.02 x 10(5)M(-1)) is more favorable than the value for GOase(ox). Effects of H+Tyr-495 deprotonation on K are smaller than those for the H2O/OH- change. The pK(1a) for GOase(semi) is very similar (5.6) to that for GOase(ox) (both at Cu-II), but pK(2a) is 8.0. At pH 7.0 values of K for GOase(semi) are NCS- (270 M-1), N-3(-) (4.9 x 10(7)), and CH3CO2- (107).