Cross-Linking Mass Spectrometry and Mutagenesis Confirm the Functional Importance of Surface Interactions between CYP3A4 and Holo/Apo Cytochrome b5.

Cytochrome b5 (cyt b5) is one of the key components in the microsomal cytochrome P450 monooxygenase system. Consensus has not been reached about the underlying mechanism of cyt b5 modulation of CYP catalysis. Both cyt b5 and apo b5 are reported to stimulate the activity of several P450 isoforms. In this study, the surface interactions of both holo and apo b5 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 b5 with CYP3A4 were built using the identified interacting sites as constraints. Both cyt b5 and apo b5 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 b5 binding affinity. The critical role of Arg446 on CYP3A4 in binding to cyt b5 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 b5 can dock with CYP3A4 in a manner analogous to that of holo b5, so electron transfer from cyt b5 is not required for its effects. [ABSTRACT FROM AUTHOR]