title: Fluoride effects on the activity of Rhus laccase and the catalytic mechanism under steady-state conditions.
abstract: Laccase uses three types of Cu(II) sites to catalyze the reduction of O2 to H2O. Fluoride binds to the type 2 site. The effects of F- on the kinetics of O2 reduction were examined to determine the catalytic roles of the copper sites. Under steady-state conditions, F- rapidly inhibits the oxidation of dimethylphenylenediamine. Both reductant-dependent and -independent steps are inhibited. Rapid-freeze ESR spectra under steady-state conditions showed that F- decreased the steady-state concentrations of oxidized type 1 copper and oxidized type 2 copper while increasing the concentration of an oxygen radical intermediate. Stopped-flow kinetic experiments were used to determine the catalytic step(s) affected by F-. The most significant effect of F- was on the reductant-dependent rate of reduction of the type 3 site. While a strictly first-order dependence was observed in the absence of F-, a hyperbolic dependence was detected in the presence of F- indicating a limiting reductant-independent step. The steady-state kinetic rapid-freeze ESR and stopped-flow kinetic data are consistent with the implicated step being the reduction of the oxygen radical in an intermediate containing reduced type 1 and reduced type 2 copper. The results suggest a role for the type 2 Cu(I) site in binding the oxygen radical and catalyzing its reduction to H2O.
authors.affiliation: Department of Biochemistry, State University of New York at Buffalo 14214.
authors.forename: G B
authors.initials: GB
authors.lastname: Koudelka
authors.forename: M J
authors.initials: MJ
authors.lastname: Ettinger
mesh_terms: D000693:Anaerobiosis; D004578:Electron Spin Resonance Spectroscopy; D005459:Fluorides / Q000494:pharmacology*; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D042845:Laccase; D010084:Oxidation-Reduction; D010088:Oxidoreductases / Q000037:antagonists & inhibitors*; D010944:Plants / Q000201:enzymology*; D010947:Plants, Toxic; D012251:Toxicodendron / Q000201:enzymology
chemical_list: D010088:Oxidoreductases; D042845:Laccase; D005459:Fluorides