Cross-Linking Mass Spectrometry and Mutagenesis Confirm the Functional Importance of Surface Interactions between CYP3A4 and Holo/Apo Cytochrome <i>b</i>₅

Cytochrome <i>b</i>₅ (cyt <i>b</i>₅) is one of the key components in the microsomal cytochrome P450 monooxygenase system. Consensus has not been reached about the underlying mechanism of cyt <i>b</i>₅ modulation of CYP catalysis. Both cyt <i>b</i>₅ and apo <i>b</i>₅ are reported to stimulate the activity of several P450 isoforms. In this study, the surface interactions of both holo and apo <i>b</i>₅ with CYP3A4 were investigated and compared for the first time. Chemical cross-linking coupled with mass spectrometric analysis was used to identify the potential electrostatic interactions between the protein surfaces. Subsequently, the models of interaction of holo/apo <i>b</i>₅ with CYP3A4 were built using the identified interacting sites as constraints. Both cyt <i>b</i>₅ and apo <i>b</i>₅ were predicted to bind to the same groove on CYP3A4 with close contacts to the B–B′ loop of CYP3A4, a substrate recognition site. Mutagenesis studies further confirmed that the interacting sites on CYP3A4 (Lys96, Lys127, and Lys421) are functionally important. Mutation of these residues reduced or abolished cyt <i>b</i>₅ binding affinity. The critical role of Arg446 on CYP3A4 in binding to cyt <i>b</i>₅ and/or cytochrome P450 reductase was also discovered. The results indicated that electrostatic interactions on the interface of the two proteins are functionally important. The results indicate that apo <i>b</i>_<i>5</i> can dock with CYP3A4 in a manner analogous to that of holo <i>b</i>₅, so electron transfer from cyt <i>b</i>₅ is not required for its effects