Electron transfer properties and catalytic competence of cytochrome b5 in the fusion protein Hmwb5-EGFP in reactions catalyzed by cytochrome P450 3A4.
In the present paper we describe studies on molecular mechanisms of protein-protein interactions between cytochrome P450 3A4 (CYP3A4) and cytochrome b(5), the latter being incorporated into the artificial recombinant protein Hmwb(5)-EGFP containing full-length cytochrome b(5) (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 b(5) within the fusion protein Hmwb(5)-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(5)-EGFP was estimated based on spin equilibrium shift of cytochrome P450 3A4 to high-spin state in the presence of Hmwb(5)-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(5)-EGFP gives an independent method to determine dissociation constant for the complex of cytochrome P450 and cytochrome b(5) 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 6beta-hydroxylation in the presence of Hmwb(5)-EGFP indicates that cytochrome b(5) in the fusion protein is able to stimulate the hydroxylation reaction. Using other fusion proteins containing either cytochrome b(5) or its hydrophilic domain to reconstitute catalytic activity of cytochrome P450 3A4 showed that the hydrophobic domain of cytochrome b(5) participates not only in hemeprotein interaction, but also in electron transfer from cytochrome b(5) to cytochrome P450.
Yantsevich
A V
AV
Institute of Bioorganic Chemistry, National Academy of Sciences of Belarus, Minsk, 220141, Belarus.
Gilep
A A
AA
Usanov
S A
SA
D000595:Amino Acid Sequence; D000818:Animals; D002384:Catalysis; D051544:Cytochrome P-450 CYP3A; D003577:Cytochrome P-450 Enzyme System / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D015786:Cytochromes b5 / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D004579:Electron Transport; D049452:Green Fluorescent Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D007700:Kinetics; D008968:Molecular Conformation; D008969:Molecular Sequence Data; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D016415:Sequence Alignment
D011993:Recombinant Fusion Proteins; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins; D015786:Cytochromes b5; D003577:Cytochrome P-450 Enzyme System; C465260:Cyp3a2 protein, rat; D051544:Cytochrome P-450 CYP3A