OBJECTIVE The mammalian mixed function oxidase (MFO) system participates in hydroxylation of many hydrophobic endogenous compounds as well as xenobiotics such as drugs and carcinogens. This biotransformation system, located in a membrane of endoplasmic reticulum, consists of cytochrome P-450 (P450), NADPH:P450 oxidoreductase and a facultative component, cytochrome b5. The knowledge of the interactions among the individual components of the MFO system is essential to understand the relationships between the structure and function of this system that finally dictate a qualitative and quantitative pattern of produced metabolites (e.g. detoxified xenobiotics and/or activated carcinogens). To elucidate the quantitative aspects of the interactions within the MFO system we acquired the photo-initiated cross-linking approach. METHODS The photo-initiated cross-linking employing cytochrome b5 as a protein nanoprobe [an amino acid analogue of methionine (pMet) was incorporated into cytochrome b5 sequence during recombinant expression] was used to quantify its interaction with P450 2B4 in a functional membrane complex. The cross-linking was initiated by UV-irradiation that formed from a pMet photolabile diazirine group highly reactive carbene biradical. This biradical is able to covalently bind amino acids in the close proximity and to form cross-link. The Met 96 of cytochrome b5 is situated in a linker region between its catalytic and membrane domains, while Met 126 and 131 are located in its membrane domain. The combination of several methods (electrophoresis in polyacrylamide gel, isoelectric focusing, Edman N-terminal degradation and amino acid analysis) was employed to characterize the molar ratio of P450 2B4 to cytochrome b5 in formed covalent cross-links to quantify their transient interactions. RESULTS The successfully produced cytochrome b5 nanoprobe (with confirmed pMet incorporation by mass spectrometry) stimulates the catalytical activity of P450 2B4 when reconstituted with NADPH:P450 oxidoreductase in vitro in dilauroylphosphatidylcholine (DLPC) vesicles. The cross-linking was carried out in similar reconstituted system without NADPH:P450 oxidoreductase, and at least three products were separated on 1D SDS-PAGE. The molar ratio of P450 to cytochrome b5 in each complex was estimated using the above-mentioned combination of methods as 1:1, 1:2 and 2:1. CONCLUSIONS The results demonstrate the utility of cytochrome b5 nanoprobe to study the interactions in MFO system. Using this nanoprobe, heterodimer with P450 2B4 and in addition also heterooligomers were identified, suggesting rather complex interactions of both proteins in this system that suppose the formation of such multimeric structures in the membrane of endoplasmic reticulum.