J. Biol. Chem., Vol. 263, Issue 8, 3698-3705, Mar, 1988
Fluoride effects on the activity of Rhus laccase and the catalytic mechanism under steady-state conditions
GB Koudelka and MJ Ettinger
Department of Biochemistry, State University of New York at Buffalo 14214.
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.
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