Conformational heterogeneity of cytochrome P450 3A4 revealed by high pressure spectroscopy.
We applied hydrostatic pressure perturbation to study substrate-induced transitions in human cytochrome P450 3A4 (CYP3A4) with bromocriptine (BCT) as a substrate. The barotropic behavior of the purified enzyme in solution was compared with that observed in recombinant microsomes of Saccharomyces cerevisiae coexpressing CYP3A4, cytochrome b(5), (b(5)) and NADPH-cytochrome P450 reductase (CPR). Important barotropic heterogeneity of CYP3A4 was detected in both cases. Only about 70% of CYP3A4 in solution and about 50% of the microsomal enzyme were susceptible to a pressure-induced P450-->P420 transition. The results suggest that both in solution and in the membrane CYP3A4 is represented by two conformers with different positions of spin equilibrium and different barotropic properties. No interconversion between these conformers was observed within the time frame of the experiment. Importantly, a pressure-induced spin shift, which is characteristic of all cytochromes P450 studied to date, was detected in CYP3A4 in solution only; the P450-->P420 transition was the sole pressure-induced process detected in microsomes. This fact suggests unusual stabilization of the high-spin state of CYP3A4, which is assumed to reflect decreased water accessibility of the heme moiety due to specific interactions of the hemoprotein with the protein partners (b(5) and CPR) and/or membrane lipids.
Davydov
Dmitri R
DR
Department of Pharmacology and Toxicology, The University of Texas Medical Branch, Galveston, TX, USA. d.davydov@UTMB.edu
Halpert
James R
JR
Renaud
Jean-Paul
JP
Hui Bon Hoa
Gaston
G
D001971:Bromocriptine / Q000737:chemistry* / Q000378:metabolism*; D051544:Cytochrome P-450 CYP3A; D003577:Cytochrome P-450 Enzyme System / Q000737:chemistry* / Q000378:metabolism*; D004789:Enzyme Activation; D004795:Enzyme Stability; D006801:Humans; D007527:Isoenzymes / Q000737:chemistry / Q000378:metabolism; D008861:Microsomes; D044367:Phase Transition; D011312:Pressure; D011485:Protein Binding; D011487:Protein Conformation; D012996:Solutions; D013057:Spectrum Analysis / Q000379:methods*; D013379:Substrate Specificity
D007527:Isoenzymes; D012996:Solutions; D001971:Bromocriptine; D003577:Cytochrome P-450 Enzyme System; C104464:CYP3A protein, human; D051544:Cytochrome P-450 CYP3A; C510163:CYP3A4 protein, human