title: Allosteric mechanisms in cytochrome P450 3A4 studied by high-pressure spectroscopy: pivotal role of substrate-induced changes in the accessibility and degree of hydration of the heme pocket.
abstract: Allosteric mechanisms in human cytochrome P450 3A4 (CYP3A4) in oligomers in solution or monomeric enzyme incorporated into Nanodiscs (CYP3A4ND) were studied by high-pressure spectroscopy. The allosteric substrates 1-pyrenebutanol (1-PB) and testosterone were compared with bromocriptine (BCT), which shows no cooperativity. In both CYP3A4 in solution and CYP3A4ND, we observed a complete pressure-induced high-to-low spin shift at pressures of <3 kbar either in the substrate-free enzyme or in the presence of BCT. In addition, both substrate-free and BCT-bound enzyme revealed a pressure-dependent equilibrium between two states with different barotropic parameters designated R for relaxed and P for pressure-promoted conformations. This pressure-induced conformational transition was also observed in the studies with 1-PB and testosterone. In CYP3A4 oligomers, the transition was accompanied by an important increase in homotropic cooperativity with both substrates. Surprisingly, at high concentrations of allosteric substrates, the amplitude of the spin shift in both CYP3A4 in solution and Nanodiscs was very low, demonstrating that hydrostatic pressure induces neither substrate dissociation nor an increase in the heme pocket hydration in the complexes of the pressure-promoted conformation of CYP3A4 with 1-PB or testosterone. These findings suggest that the mechanisms of interactions of CYP3A4 with 1-PB and testosterone involve an effector-induced transition that displaces a system of conformational equilibria in the enzyme toward the state(s) with decreased solvent accessibility of the active site so that the flux of water into the heme pocket is impeded and the high-spin state of the heme iron is stabilized.
authors.affiliation: Department of Pharmacology and Toxicology, The University of Texas Medical Branch, 301 University Boulevard, Galveston, Texas 77555, USA. d.davydov@utmb.edu
authors.forename: Dmitri R
authors.initials: DR
authors.lastname: Davydov
authors.forename: Bradley J
authors.initials: BJ
authors.lastname: Baas
authors.forename: Stephen G
authors.initials: SG
authors.lastname: Sligar
authors.forename: James R
authors.initials: JR
authors.lastname: Halpert
mesh_terms: D000494:Allosteric Regulation / Q000502:physiology*; D001971:Bromocriptine / Q000494:pharmacology; D051544:Cytochrome P-450 CYP3A; D003577:Cytochrome P-450 Enzyme System / Q000737:chemistry* / Q000378:metabolism; D004578:Electron Spin Resonance Spectroscopy; D006418:Heme / Q000737:chemistry*; D006874:Hydrostatic Pressure; D036103:Nanotechnology; D025341:Principal Component Analysis; D011487:Protein Conformation; D011721:Pyrenes / Q000378:metabolism / Q000494:pharmacology; D013056:Spectrophotometry, Ultraviolet; D013739:Testosterone / Q000378:metabolism / Q000494:pharmacology
chemical_list: C463480:1-pyrenebutanol; D011721:Pyrenes; D001971:Bromocriptine; D013739:Testosterone; D006418:Heme; D003577:Cytochrome P-450 Enzyme System; D051544:Cytochrome P-450 CYP3A; C510163:CYP3A4 protein, human