| Abstract
| - NADP(H) binding is essential for fast electron transfer through the flavoprotein domain ofthe fusion protein P450BM3. Here we characterize the interaction of NADP(H) with the oxidized andpartially reduced enzyme and the effect of this interaction on the redox properties of flavin cofactors andelectron transfer. Measurements by three different approaches demonstrated a relatively low affinity ofoxidized P450BM3 for NADP+, with a Kd of about 10 μM. NADPH binding is also relatively weak (Kd∼10 μM), but the affinity increases manyfold upon hydride ion transfer so that the active 2-electronreduced enzyme binds NADP+ with a Kd in the submicromolar range. NADP(H) binding inducesconformational changes of the protein as demonstrated by tryptophan fluorescence quenching. Fluorescencequenching indicated preferential binding of NADPH by oxidized P450BM3, while no catalyticallycompetent binding with reduced P450BM3 could be detected. The hydride ion transfer step, as well asthe interflavin electron transfer steps, is readily reversible, as demonstrated by a hydride ion exchange(transhydrogenase) reaction between NADPH and NADP+ or their analogues. Experiments with FMN-free mutants demonstrated that FAD is the only flavin cofactor required for the transhydrogenase activity.The equilibrium constants of each electron transfer step of the flavoprotein domain during catalytic turnoverhave been calculated. The values obtained differ from those calculated from equilibrium redox potentialsby as much as 2 orders of magnitude. The differences result from the enzyme's interaction with NADP(H).
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