Understanding uncoupling in the multiredox centre P450 3A4-BMR model system.
Understanding the uncoupling at the haem active site and/or at the level of multidomain electron transfer is an important element in cytochrome P450 chemistry. Here a chimeric model system consisting of human cytochrome P450 3A4 and the soluble reductase domain of CYP102A1 from Bacillus megaterium (BMR) is used to study the relationship between electron transfer and the coupling efficiency in substrate monoxygenation. Several regulatory features were considered. FAD and FMN added to apoenzyme in oversaturating concentrations influence neither formaldehyde production nor coupling efficiency. The optimal conditions of coupling efficiency depended only on the NADPH concentration. The pH (8.0) and ionic strength (50 mM potassium phosphate) were found to modulate the level of coupling, indicating an influence over the formation of a productive interaction between the BMR and the haem domain. Overall, uncoupling is found to be an intrinsic property of the haem domain, and the covalent linkage of the reductase in a single polypeptide chain has little influence over the activity coupled to product formation.
Degregorio
Danilo
D
Department of Human and Animal Biology, University of Turin, Via Accademia Albertina 13, Turin, Italy.
Sadeghi
Sheila J
SJ
Di Nardo
Giovanna
G
Gilardi
Gianfranco
G
Solinas
Sandro P
SP
D001426:Bacterial Proteins / Q000737:chemistry / Q000378:metabolism*; D051544:Cytochrome P-450 CYP3A / Q000737:chemistry / Q000378:metabolism*; D003577:Cytochrome P-450 Enzyme System / Q000737:chemistry / Q000378:metabolism*; D004579:Electron Transport; D006418:Heme / Q000737:chemistry / Q000378:metabolism; D006801:Humans; D006863:Hydrogen-Ion Concentration; D008954:Models, Biological; D009249:NADP / Q000737:chemistry / Q000378:metabolism; D009251:NADPH-Ferrihemoprotein Reductase / Q000737:chemistry / Q000378:metabolism*; D009994:Osmolar Concentration; D010084:Oxidation-Reduction; D010088:Oxidoreductases / Q000737:chemistry / Q000378:metabolism; D010710:Phosphates / Q000737:chemistry; D017680:Potassium Compounds / Q000737:chemistry; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism
D001426:Bacterial Proteins; D010710:Phosphates; D017680:Potassium Compounds; D011994:Recombinant Proteins; D006418:Heme; D009249:NADP; D003577:Cytochrome P-450 Enzyme System; C013216:potassium phosphate; D010088:Oxidoreductases; D051544:Cytochrome P-450 CYP3A; D009251:NADPH-Ferrihemoprotein Reductase; C052423:flavocytochrome P450 BM3 monoxygenases