Effects of a Single Reduction-Reoxidation Cycle on the Kinetics of Copper Reductions in Rhus Laccase

Absorbance and fluorescence stopped-flow kinetic experiments were designed to examine the effects that catalytic turnover has on Rhus laccase kinetics. Biphasic type 3 Cu reduction kinetics are observed under anaerobic reducing conditions at pH 7.4 or 8.5. This suggests kinetically active and inactive oxidized enzyme forms. Inhibited type 3 reduction phases are abolished by a single reduction-reoxidation cycle. The kinetic profiles associated with types 1 and 3 Cu rereduction and the concurrent fluorescence enhancement all slowly return to those exhibited by the resting enzyme as rereduction is initiated at increasing times after the single turnover. The calculated rate of this transformation from activated to resting enzyme equals the rate of type 2 Cu(I) oxidation when minimally reduced enzyme is mixed with 02. In contrast, the rate of type 2 Cu(I) oxidation is much faster when excess reductant is present during oxidation by 02 as expected if the type 2 Cu(I) has a role in the reduction of the type 3 copper pair. The results are consistent with a steady-state kinetic reaction sequence in which the unliganded, fully oxidized enzyme does not participate. A pivotal role for an intermediate in which the type 2 Cu is reduced is proposed. To satisfactorily account for the 30% reduced types 1 and 2 Cu(I) during steady-state turnover, it is also suggested that reduction of the oxygen radical intermediate occurs only after reduction of both type 1 and type 2 Cu(II).