[Objective] Cytochrome P450 (CYP) enzymes are important contributors to xenobiotic metabolism, among which the CYP3A4 and CYP2 family account for ~80% of the total liver P450s. Cytochrome b5 (cyt b5 or holo b5) interacts with many CYP isoforms and can markedly modulate their activities. The effect is both substrate- and CYP-dependent, either increasing, decreasing or having no effect on CYP activities. In addition, cyt b5 devoid of heme (apo b5) can stimulate the activities of some CYP isoforms (3A4, 2A6, 2C9) to a similar extent as holo b5, but not the activities of other CYP isoforms (2E1, 2D6). The underlying molecular mechanism for this differential modulation has not yet been determined. The current investigation was focused on the surface interactions of cyt b5 with various CYP isoforms as possible contributing factors in the differential modulation. [Methods] Recombinant human CYPs (3A4, 2A6, 2C9, 2D6 and 2E1) were each reconstituted with cyt b5. Interacting sites between CYP isoforms and cyt b5 were identified using the zero-linker cross-linking reagent 1-ethyl-3-[3-dimethylaminopropyl]carbodiimide hydrochloride (EDC), coupled with mass spectrometric analysis. Computer models of their interactions were built using the cross-linked sites as constraints in addition to the crystal structures of individual CYPs and a homology model of human cyt b5. Interactions between apo b5 and CYPs were also investigated using the same methods. In order to confirm the biological significance of the amino acid residues on CYP3A4 involved in cross-linking with cyt b5, site-directed mutagenesis was carried out followed by reassessment of CYP3A4 activities using Vivid Green and testosterone as probe substrates. [Results] Cross-linking sites between CYPs and holo b5/apo b5 were identified, and computer models of their interactions were developed. The heme in holo cyt b5 and in CYP2A6/2C9/3A4 are not as closely associated as in the published interaction model of holo b5-CYP2E1, indicating holo b5 stimulates the activities of these three CYPs differently than CYP2E1. The single or triple mutations of cross-linked residues (K96A, K127A, K421A) on CYP3A4 decreased its activity to various degrees. The conserved amino acid R446 was predicted to be critical in the interaction between cyt b5 and CYP3A4 based on a holo b5-CYP3A4 interaction model. Mutation at this position R446A abolished the testosterone 6-beta hydroxylation and Vivid Green metabolism activity of CYP3A4. Overall, the data indicates that sites on CYPs that interact with cyt b5, as identified by chemical cross-linking, likely play important roles in the activities of some CYP isoforms. [Conclusion] The present study indicates that cyt b5 modulates CYP2A6/2C9/3A4 activities without direct electron transfer. This is a different mechanism than that proposed for the interaction of cyt b5 with CYP2E1, which likely involves electron transfer between the heme groups of these two cytochromes. Acknowledgment: Supported by NIH/NIGMS Program Project Grant No. GM32165 and the Univerisity of Washington Proteomics Resource center Grant No. UWPR95794. We thank Dr. David R. Goodlett and Dr. Priska von Haller for their assistance with the proteomics analysis, and the Collaboratory for MS3D Portal.