Kinetics of electron transfer in the complex of cytochrome P450 3A4 with the flavin domain of cytochrome P450BM-3 as evidence of functional heterogeneity of the heme protein.
We used a rapid scanning stop-flow technique to study the kinetics of reduction of cytochrome P450 3A4 (CYP3A4) by the flavin domain of cytochrome P450-BM3 (BMR), which was shown to form a stoichiometric complex (K(D)=0.48 microM) with CYP3A4. In the absence of substrates only about 50% of CYP3A4 was able to accept electrons from BMR. Whereas the high-spin fraction was completely reducible, the reducibility of the low-spin fraction did not exceed 42%. Among four substrates tested (testosterone, 1-pyrenebutanol, bromocriptine, or alpha-naphthoflavone (ANF)) only ANF is capable of increasing the reducibility of the low-spin fraction to 75%. Our results demonstrate that the pool of CYP3A4 is heterogeneous, and not all P450 is competent for electron transfer in the complex with reductase. The increase in the reducibility of the enzyme in the presence of ANF may represent an important element of the mechanism of action of this activator.
Fernando
Harshica
H
Department of Pharmacology and Toxicology, The University of Texas Medical Branch, 301 University Boulevard, Galveston, TX 77555-1031, United States.
Halpert
James R
JR
Davydov
Dmitri R
DR
D000494:Allosteric Regulation; D001426:Bacterial Proteins / Q000737:chemistry* / Q000378:metabolism*; D001571:Benzoflavones / Q000737:chemistry; D003577:Cytochrome P-450 Enzyme System / Q000737:chemistry* / Q000378:metabolism*; D004579:Electron Transport; D029968:Escherichia coli Proteins / Q000737:chemistry* / Q000378:metabolism*; D005415:Flavins / Q000737:chemistry* / Q000378:metabolism; D006420:Hemeproteins / Q000502:physiology*; D007700:Kinetics; D006899:Mixed Function Oxygenases / Q000737:chemistry* / Q000378:metabolism*; D009251:NADPH-Ferrihemoprotein Reductase; D010084:Oxidation-Reduction; D017434:Protein Structure, Tertiary; D013379:Substrate Specificity
D001426:Bacterial Proteins; D001571:Benzoflavones; D029968:Escherichia coli Proteins; D005415:Flavins; D006420:Hemeproteins; C011512:alpha-naphthoflavone; D003577:Cytochrome P-450 Enzyme System; D006899:Mixed Function Oxygenases; D009251:NADPH-Ferrihemoprotein Reductase; C052423:flavocytochrome P450 BM3 monoxygenases