Energetics of heterotropic cooperativity between α-naphthoflavone and testosterone binding to CYP3A4

Cytochrome P450 3A4 (CYP3A4) is involved in the metabolism of a majority of drugs. Heterotropic cooperativity of drug binding to CYP3A4 was examined with the flavanoid, α-naphthoflavone (ANF) and the steroid, testosterone (TST). UV–vis and EPR spectroscopy of CYP3A4 show that ANF binding to CYP3A4 occurs with apparent negative cooperativity and that there are at least two binding sites: (1) a relatively tight spin-state insensitive binding site (CYP·ANF) and (2) a relatively low affinity spin-state sensitive binding site (CYP·ANF·ANF). Since binding to the spin-state insensitive binding site is considerably tighter for ANF than TST, the spin-state insensitive binding site could be occupied by ANF, while titrating TST at the other site(s). The spin-state insensitive binding site of ANF appears to compete with the spin-state insensitive binding site of TST. The formation of the spin-state insensitive CYP·ANF complex is strongly temperature dependent, when compared to the formation of the CYP·TST complex, suggesting that the formation of the CYP3A4·ANF complex leads to long-range conformational changes within the protein. When the CYP·ANF complex is titrated with TST, the formation of CYP·ANF·TST is favored by 3:1 over the formation of CYP·TST·TST, suggesting that there is an allosteric interaction between ANF and TST. A model of heterotropic cooperativity of CYP3A4 is presented, where the spin-state insensitive binding of ANF occurs at the same peripheral binding site of CYP3A4 as TST.