Absorbance and Fluorescence Stopped-Flow Kinetics of Rhus Lacease and the Catalytic Reaction Sequence

Anaerobic reduction of Rhus lacease causes a 1.76-fold increase in protein fluorescence [Goldberg, M., & Pecht, I. (1974) Proc. Natl. Acad. Sci. U.S.A. 71, 4684], Parallel stopped-flow kinetic absorbance and fluorescence measurements were made to determine with which reduction step(s) the fluorescence changes were associated. Anaerobic reduction data obtained at pH 6.0, 7.4, and 8.5 with and without fluoride showed that fluorescence increases are either slower than (e.g., pH 6.0, 0.25 mM ascorbate) or faster than [e.g., pH 6.0, 0.1 mM Ru(NH₃)₆ ²⁺] net type 1 Cu(II) reduction. Fluorescence increases are usually slower than type 3 reduction. However, type 3 reduction and fluorescence enhancement profiles are congruent in the presence of fluoride or at pH 8.5; the fluorescence changes are never faster than type 3 reduction. The forms of the fluorescence kinetic profiles invariably are similar to the type 3 reduction profiles and not the type 1. The oxidation of reduced copper sites by 02 and the associated fluorescence decrease occur with the same rate constant. Satisfactory simulations are only obtained by assuming that two fluorescence changes occur which are associated with two separate reduction steps. The data are consistent with a first fluorescence increase occurring upon type 3 reduction and a second with the final type 1 Cu(II) reduction. This suggests that a change in protein conformation occurs when the type 3 site is reduced which in turn makes fluorescence sensitive to the redox state of the type 1 copper. pH-jump experiments indicate that the active and inactive forms evident at pH 7.4 and 8.5 [Andreasson, L.-E., & Reinhammar, B. (1976) Biochim. Biophys. Acta 445, 579] are not in equilibrium in the fully oxidized enzyme.