Electron Transfer Properties and Catalytic Competence of Cytochrome b5 in the Fusion Protein Hmwb5-EGFP in Reactions Catalyzed by Cytochrome P450 3A4

摘要

In the present paper we describe studies on molecular mechanisms of protein-protein interactions between cytochrome P450 3A4 (CYP3A4) and cytochrome b5, the latter being incorporated into the artificial recombinant protein Hmwb5-EGFP containing full-length cytochrome b5 (functional module) and a mutant form of the green fluorescent protein EGFP (signal module) fused into a single polypeptide chain. It is shown that cytochrome b5 within the fusion protein Hmwb5-EGFP can be reduced by NADPH-cytochrome P450 reductase in the presence of NADPH, the rate of reduction being dependent on solution ionic strength, indicating that the signal module does not prevent the interaction of the flavo-and hemeproteins. Interaction of cytochrome P450 3A4 and Hmwb-EGFP was estimated based on spin equilibrium shift of cytochrome P450 3A4 to high-spin state in the presence of Hmwb-EGFP, as well as based on steady-state fluorescence anisotropy of the EGFP component of the fusion protein in the presence of CYP3A4. The engineering of chimeric protein Hmwb-EGFP gives an independent method to determine dissociation constant for the complex of cytochrome P450 and cytochrome b5 that is less sensitive to environmental factors compared to spectrophotometric titration used before. Reconstitution of catalytic activity of cytochrome P450 3A4 in the reaction of testosterone 6p-hydroxylation in the presence of Hmwb-EGFP indicates that cytochrome b5 in the fusion protein is able to stimulate the hydroxylation reaction. Using other fusion proteins containing either cytochrome b5 or its hydrophilic domain to reconstitute catalytic activity of cytochrome P450 3A4 showed that the hydrophobic domain of cytochrome b5participates not only in herneprotein interaction, but also in electron transfer from cytochrome b5 to cytochrome P450.