The one-electron autoxidation of human cytochrome P450 3A4.
Monomeric cytochrome P450 3A4 (CYP3A4), the most prevalent cytochrome P450 in human liver, can simultaneously bind one, two, or three molecules of substrates and effectors. The difference in the functional properties of such binding intermediates gives rise to homotropic and heterotropic cooperative kinetics of this enzyme. To understand the overall kinetic processes operating in CYP3A4, we documented the kinetics of autoxidation of the oxy-ferrous intermediate of CYP3A4 as a function of testosterone concentration. The rate of autoxidation in the presence of testosterone was significantly lower than that observed with no substrate present. Stability of the oxy-ferrous complex in CYP3A4 and the amplitude of the geminate CO rebinding increased significantly as a result of binding of just one testosterone molecule. In contrast, the slow phase in the kinetics of cyanide binding to the ferric CYP3A4 correlated with a shift of the heme iron spin state, which is only caused by the association of a second molecule of testosterone. Our results show that the first substrate binding event prevents the escape of diatomic ligands from the distal heme binding pocket, stabilizes the oxy-ferrous complex, and thus serves as an important modulator of the uncoupling channel in the cytochromes P450.
Denisov
Ilia G
IG
Departments of Biochemistry, University of Illinois, Urbana, Illinois 61801.
Grinkova
Yelena V
YV
Departments of Biochemistry, University of Illinois, Urbana, Illinois 61801.
McLean
Mark A
MA
Departments of Biochemistry, University of Illinois, Urbana, Illinois 61801.
Sligar
Stephen G
SG
Departments of Biochemistry, University of Illinois, Urbana, Illinois 61801; Departments of Chemistry, University of Illinois, Urbana, Illinois 61801; Center for Biophysics and Computational Biology, University of Illinois, Urbana, Illinois 61801; College of Medicine, University of Illinois, Urbana, Illinois 61801. Electronic address: s-sligar@uiuc.edu.
D002248:Carbon Monoxide / Q000378:metabolism; D003486:Cyanides / Q000378:metabolism; D051544:Cytochrome P-450 CYP3A; D003577:Cytochrome P-450 Enzyme System / Q000737:chemistry*; D004583:Electrons; D006801:Humans; D010084:Oxidation-Reduction; D013739:Testosterone / Q000378:metabolism
D003486:Cyanides; D013739:Testosterone; D002248:Carbon Monoxide; D003577:Cytochrome P-450 Enzyme System; D051544:Cytochrome P-450 CYP3A; C510163:CYP3A4 protein, human