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

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A. V. Yantsevich, A. A. Gilep, and S. A. Usanov^*

Institute of Bioorganic Chemistry, National Academy of Sciences of Belarus, ul. Kuprevicha 5/2, 220141 Minsk, Belarus; fax: 375 (172) 63-7274; E-mail: usanov@iboch.bas-net.by

^* To whom correspondence should be addressed.

Received January 27, 2009; Revision received February 19, 2009

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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 Hmwb₅–EGFP was estimated based on spin equilibrium shift of cytochrome P450 3A4 to high-spin state in the presence of Hmwb₅–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 Hmwb₅–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 6β-hydroxylation in the presence of Hmwb₅–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 b₅ participates not only in hemeprotein interaction, but also in electron transfer from cytochrome b₅ to cytochrome P450.

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KEY WORDS: microsomal cytochrome b₅, green fluorescent protein, cytochrome P450 3A4, testosterone 6β-hydroxylation

DOI: 10.1134/S0006297909080070

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