7,8-benzoflavone binding to human cytochrome P450 3A4 reveals complex fluorescence quenching, suggesting binding at multiple protein sites

<p>Human cytochrome P450 (P450) 3A4 is involved in the metabolism of one-half of marketed drugs and shows cooperative interactions with some substrates and other ligands. The interaction between P450 3A4 and the known allosteric effector 7,8-benzoflavone (<i>α</i>-naphthoflavone, <i>α</i>NF) was characterized using steady-state fluorescence spectroscopy. The binding interaction of P450 3A4 and <i>α</i>NF effectively quenched the fluorescence of both the enzyme and ligand. The Hill Equation and Stern–Volmer fluorescence quenching models were used to evaluate binding of ligand to enzyme. P450 3A4 fluorescence was quenched by titration with <i>α</i>NF; at the relatively higher [<i>α</i>NF]/[P450 3A4] ratios in this experiment, two weaker quenching interactions were revealed (<i>K</i><sub><i>d</i></sub> 1.8–2.5 and 6.5 μM). A range is given for the stronger interaction since <i>α</i>NF quenching of P450 3A4 fluorescence changed the protein spectral profile: quenching of 315 nm emission was slightly more efficient (<i>K</i><sub><i>d</i></sub> 1.8 μM) than the quenching of protein fluorescence at 335 and 355 nm (<i>K</i><sub><i>d</i></sub> 2.5 and 2.1 μM, respectively). In the reverse titration, <i>α</i>NF fluorescence was quenched by P450 3A4; at the lower [<i>α</i>NF]/[P450 3A4] ratios here, two strong quenching interactions were revealed (<i>K</i><sub><i>d</i></sub> 0.048 and 1.0 μM). Thus, four binding interactions of <i>α</i>NF to P450 3A4 are suggested by this study, one of which may be newly recognized and which could affect studies of drug oxidations by this important enzyme.</p>