Functional interactions in cytochrome P450(BM3). Evidence that NADP(H) binding controls redox potentials of the flavin cofactors

NADP(H) binding is essential for fast electron transfer through the flavopr otein domain of the fusion protein P450BM3. Here we characterize the intera ction of NADP(H) with the oxidized and partially reduced enzyme and the eff ect of this interaction on the redox properties of flavin cofactors and ele ctron transfer. Measurements by three different approaches demonstrated a r elatively low affinity of oxidized P450BM3 for NADP(+), with a K-d Of about 10 muM. NADPH binding is also relatively weak (K-d similar to 10 muM), but the affinity increases manyfold upon hydride ion transfer so that the acti ve 2-electron reduced enzyme binds NADP(+) with a Kd in the submicromolar r ange. NADP(H) binding induces conformational changes of the protein as demo nstrated by tryptophan fluorescence quenching. Fluorescence quenching indic ated preferential binding of NADPH by oxidized P450BM3, while no catalytica lly competent binding with reduced P450BM3 could be detected. The hydride i on transfer step, as well as the interflavin electron transfer steps, is re adily reversible, as demonstrated by a hydride ion exchange (transhydrogena se) reaction between NADPH and NADP(+) or their analogues. Experiments with FMN-free mutants demonstrated that FAD is the only flavin cofactor require d for the transhydrogenase activity. The equilibrium constants of each elec tron transfer step of the flavoprotein domain during catalytic turnover hav e been calculated. The values obtained differ from those calculated from eq uilibrium redox potentials by as much as 2 orders of magnitude. The differe nces result from the enzyme's interaction with NADP(H).