Allosteric transitions in cytochrome P450eryF explored with pressure-perturbation spectroscopy, lifetime FRET, and a novel fluorescent substrate, Fluorol-7GA.
To establish a direct method for monitoring substrate binding in cytochrome P450eryF applicable at elevated hydrostatic pressures, we introduce a laser dye Fluorol-7GA (F7GA) as a novel fluorescent ligand. The high intensity of fluorescence and the reasonable resolution of the excitation band from the absorbance bands of P450 allowed us to establish highly sensitive binding assays compatible with pressure perturbation. The interactions of F7GA with P450eryF cause an ample spin shift revealing cooperative binding ( S50 = 8.2 +/- 1.3 microM; n = 2.3 +/- 0.1). Fluorescence resonance energy transfer (FRET) experiments suggest the presence of at least two substrate binding sites with apparent K D values in the ranges of 0.1-0.3 and 6-9 microM. Similar to that observed earlier with CYP3A4, increasing hydrostatic pressure does not cause either a complete dissociation of the substrate complexes or a displacement of the spin equilibrium toward the low-spin state. Rather, increased pressure enhances the cooperativity of the F7GA-induced spin shift, so that the Hill coefficient approaches 3 at 2 kbar. Lifetime FRET experiments revealed an important increase in the affinity of the enzyme for F7GA at elevated pressures, suggesting that the binding of the ligand induces a conformational transition associated with an important increase in the level of protein hydration. This transition largely attenuates the solvent accessibility of the heme pocket and causes an unusual stability of the high-spin, substrate-bound enzyme at elevated pressures.
Davydov
Dmitri R
DR
Skaggs School of Pharmacy and Pharmaceutical Sciences, University of California at San Diego, 9500 Gilman Drive, La Jolla, California 92093-0703, USA. ddavydov@ucsd.edu
Davydova
Nadezhda Y
NY
Halpert
James R
JR
D000495:Allosteric Site; D001426:Bacterial Proteins; D051544:Cytochrome P-450 CYP3A; D003577:Cytochrome P-450 Enzyme System / Q000737:chemistry* / Q000378:metabolism*; D031541:Fluorescence Resonance Energy Transfer; D005456:Fluorescent Dyes / Q000737:chemistry / Q000378:metabolism*; D006874:Hydrostatic Pressure; D007546:Isoquinolines / Q000737:chemistry / Q000378:metabolism; D007700:Kinetics; D008024:Ligands; D015394:Molecular Structure; D013050:Spectrometry, Fluorescence; D013379:Substrate Specificity; D017186:Titrimetry
C532907:2-butyl-6-(butylamino)-1H-benzo(de)isoquinoline-1,3(2H)-dione; D001426:Bacterial Proteins; D005456:Fluorescent Dyes; D007546:Isoquinolines; D008024:Ligands; D003577:Cytochrome P-450 Enzyme System; C054444:eryF protein, Saccharopolyspora erythraea; D051544:Cytochrome P-450 CYP3A; C510163:CYP3A4 protein, human