flavin-adenine-dinucleotide and 1-4-5-6-tetrahydronicotinamide-adenine-dinucleotide

NADP(H) binding is essential for fast electron transfer through the flavoprotein domain of the fusion protein P450BM3. Here we characterize the interaction of NADP(H) with the oxidized and partially reduced enzyme and the effect of this interaction on the redox properties of flavin cofactors and electron transfer. Measurements by three different approaches demonstrated a relatively low affinity of oxidized P450BM3 for NADP(+), with a K(d) of about 10 microM. NADPH binding is also relatively weak (K(d) approximately 10 microM), but the affinity increases manyfold upon hydride ion transfer so that the active 2-electron reduced enzyme binds NADP(+) with a K(d) in the submicromolar range. NADP(H) binding induces conformational changes of the protein as demonstrated by tryptophan fluorescence quenching. Fluorescence quenching indicated preferential binding of NADPH by oxidized P450BM3, while no catalytically competent binding with reduced P450BM3 could be detected. The hydride ion transfer step, as well as the 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 turnover have been calculated. The values obtained differ from those calculated from equilibrium redox potentials by as much as 2 orders of magnitude. The differences result from the enzyme's interaction with NADP(H).

Topics: Animals; Bacterial Proteins; Binding, Competitive; Cytochrome P-450 Enzyme System; Electron Transport; Flavin Mononucleotide; Flavin-Adenine Dinucleotide; Flavins; Flavoproteins; Mixed Function Oxygenases; Mutagenesis, Site-Directed; NAD; NADP; NADP Transhydrogenases; NADPH-Ferrihemoprotein Reductase; Oxidation-Reduction; Phosphorus Radioisotopes; Protein Binding; Protein Structure, Tertiary; Protons; Rats; Spectrometry, Fluorescence; Tryptophan

Topics: Electron Transport; Flavin-Adenine Dinucleotide; Glutathione Reductase; Kinetics; NAD; NADP; Oscillometry; Oxidation-Reduction; Saccharomyces cerevisiae; Spectrophotometry