No abstract

No 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.

Comparisons (25 degrees C) are made of substitution reactions, X replacing H(2)O, at the tetrahedral Ni of the heterometallic sulfido cuboidal cluster [Mo(3)NiS(4)(H(2)O)(10)](4+), I = 2.00 M (LiClO(4)). Stopped-flow formation rate constants (k(f)/M(-)(1) s(-)(1)) for six X reagents, including two water soluble air-stable phosphines, 1,3,5-triaza-7-phosphaadamantane PTA (119) and tris(3-sulfonatophenyl)phosphine TPPTS(3)(-) (58), and CO (0.66), Br(-) (14.6), I(-) (32.3), and NCS(-) (44) are reported alongside the previous value for Cl(-) (9.4). A dependence on [H(+)] is observed with PTA, which gives an unreactive form confirmed by NMR as N-protonated PTA (acid dissociation constant K(a) = 0.61 M), but in no other cases with [H(+)] in the range 0.30-2.00 M. The narrow spread of rate constants for all but the CO reaction is consistent with an I(d) dissociative interchange mechanism. In addition NMR studies with H(2)(17)O enriched solvent are too slow for direct determination of the water-exchange rate constant indicating a value <10(3) s(-)(1). Equilibrium constants/M(-)(1) for 1:1 complexing with the different X groups at the Ni are obtained for PTA (2040) and TPPTS(3)(-) (8900) by direct spectrophotometry and from kinetic studies (k(f)/k(b)) for Cl(-) (97), Br(-) (150), NCS(-) (690), and CO (5150). No NCS(-) substitution at the Ni is observed in the case of the heterometallic cube [Mo(3)Ni(L)S(4)(H(2)O)(9)](4+), with tridentate 1,4,7-triazacyclononane(L) coordinated to the Ni. Substitution of NCS(-) for H(2)O, at the Mo's of [Mo(3)NiS(4)(H(2)O)(10)](4+) and [Mo(3)(NiL)S(4)(H(2)O)(9)](4+) are much slower secondary processes, with k(f) = 2.7 x 10(-)(4) M(-)(1) s(-)(1) and 0.94 x 10(-)(4) M(-)(1) s(-)(1) respectively. No substitution of H(2)O by TPPTS(3)(-) or CO is observed over approximately 1h at either metal on [Mo(3)FeS(4)(H(2)O)(10)](4+), on [Mo(4)S(4)(H(2)O)(12)](5+) or [Mo(3)S(4)(H(2)O)(9)](4+).