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

Abstract

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 (α-naphthoflavone, αNF) was characterized using steady-state fluorescence spectroscopy. The binding interaction of P450 3A4 and αNF 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 αNF; at the relatively higher [αNF]/[P450 3A4] ratios in this experiment, two weaker quenching interactions were revealed (K_d 1.8–2.5 and 6.5 μM). A range is given for the stronger interaction since αNF quenching of P450 3A4 fluorescence changed the protein spectral profile: quenching of 315 nm emission was slightly more efficient (K_d 1.8 μM) than the quenching of protein fluorescence at 335 and 355 nm (K_d 2.5 and 2.1 μM, respectively). In the reverse titration, αNF fluorescence was quenched by P450 3A4; at the lower [αNF]/[P450 3A4] ratios here, two strong quenching interactions were revealed (K_d 0.048 and 1.0 μM). Thus, four binding interactions of αNF 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.

Keywords:

• P450 3A4
• α-naphthoflavone
• fluorescence quenching
• cytochromes P450

Acknowledgment

The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health. We thank M. V. Martin and L. M. Folkmann for preparing P450 3A4 and Thanh T. Nguyen Phan for acquiring absorption spectra of P450 3A4 in the presence of testosterone and αNF. The authors also thank many students who have assisted in this work, including Joel Fern and Dawn Akridge (Union University) and Judah Henry, Joshua Inks, David Marsch, Jon Guglielmon, David Smith, Jennifer Hansen, Elaine Mihelc, Emily Kozlowski, Becky Kraft, David Lewis, Mike Benich, Jimmy Gregory, Jeremy Smith, Tim Rogers, Samuel Niehaus, and Tucker Sigourney (Grove City College). Thanks are also extended to Grove City College for a sabbatical leave to Vanderbilt University.