Roles of divalent metal ions in oxidations catalyzed by recombinant cytochrome P450 3A4 and replacement of NADPH--cytochrome P450 reductase with other flavoproteins, ferredoxin, and oxygen surrogates.

Yamazaki H, Ueng YF, Shimada T, Guengerich FP
Biochemistry. 1995 34 (26): 8380-9

PMID: 7599128 · DOI:10.1021/bi00026a020

Recombinant cytochrome P450 (P450) 3A4 was most active in nifedipine and testosterone oxidation in a system including NADPH-P450 reductase, cytochrome b5 (b5), a semisynthetic phospholipid mixture plus cholate, glutathione, and MgCl2. The MgCl2 effect could be seen with high concentrations of Ca2+ or Sr2+ but not readily when these cations were replaced with monovalent cations. The divalent cation effect was also seen in liver microsomes. Part of the basis of this effect appears to be enhanced rates of b5 reduction, as judged from studies on deletions of reconstitution components and analysis of steady-state spectral studies. Rapid reduction of ferric P450 3A4 to ferrous was dependent upon the presence of substrate, either testosterone or ethylmorphine. When testosterone was present, reduction was also highly dependent upon the presence of b5 and Mg2+. In the case of the substrate ethylmorphine, the need to add b5 and Mg2+ to obtain optimal reduction rates was less pronounced. These patterns are consistent with the dramatic dependence of testosterone 6 beta-hydroxylation on b5 and the lack of dependence of ethylmorphine N-demethylation on b5. Our interpretation is that divalent cations stimulate electron transfer from NADPH-P450 reductase to several acceptors and that substrates and b5 can bind to P450 3A4 to influence its rate of reduction by the reductase. P450 3A4 catalyzed testosterone 6 beta-hydroxylation within Escherichia coli cells. The reactions could be supported by E. coli cytosol or by purified E. coli flavodoxin and NADPH-flavodoxin reductase. Spinach ferredoxin and NADPH-ferredoxin reductase also supported catalytic activities.(ABSTRACT TRUNCATED AT 250 WORDS)

MeSH Terms (22)

Animals Calcium Chloride Catalysis Cations, Divalent Cytochrome P-450 CYP3A Cytochrome P-450 Enzyme System Escherichia coli Ethylmorphine Homeostasis Kinetics Magnesium Chloride Microsomes, Liver Mixed Function Oxygenases Models, Biological NADPH-Ferrihemoprotein Reductase Nifedipine Oxidation-Reduction Rabbits Recombinant Proteins Spectrophotometry Spinacia oleracea Testosterone

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