Spectroscopic characterization of the oxyferrous complex of prostacyclin synthase in solution and in trapped sol-gel matrix.
Prostacyclin synthase (PGIS) is a member of the cytochrome P450 family in which the oxyferrous complexes are generally labile in the absence of substrate. At 4 degrees C, the on-rate constants and off-rate constants of oxygen binding to PGIS in solution are 5.9 x 10(5) m(-1).s(-1) and 29 s(-1), respectively. The oxyferrous complex decays to a ferric form at a rate of 12 s(-1). We report, for the first time, a stable oxyferrous complex of PGIS in a transparent sol-gel monolith. The encapsulated ferric PGIS retained the same spectroscopic features as in solution. The binding capabilities of the encapsulated PGIS were demonstrated by spectral changes upon the addition of O-based, N-based and C-based ligands. The peroxidase activity of PGIS in sol-gel was three orders of magnitude slower than that in solution owing to the restricted diffusion of the substrate in sol-gel. The oxyferrous complex in sol-gel was observable for 24 h at room temperature and displayed a much red-shifted Soret peak. Stabilization of the ferrous-carbon monoxide complex in sol-gel was observed as an enrichment of the 450-nm species over the 420-nm species. This result suggests that the sol-gel method may be applied to other P450s to generate a stable intermediate in the di-oxygen activation.
Yeh
Hui-Chun
HC
Division of Hematology, Department of Internal Medicine, University of Texas Health Science Center, Houston, TX 77030, USA.
Hsu
Pei-Yung
PY
Tsai
Ah-Lim
AL
Wang
Lee-Ho
LH
D003577:Cytochrome P-450 Enzyme System / Q000235:genetics / Q000378:metabolism*; D005109:Extracellular Matrix / Q000737:chemistry*; D005296:Ferrous Compounds / Q000737:chemistry* / Q000378:metabolism; D019746:Intramolecular Oxidoreductases / Q000235:genetics / Q000378:metabolism*; D007700:Kinetics; D008024:Ligands; D010084:Oxidation-Reduction; D010100:Oxygen / Q000737:chemistry / Q000378:metabolism; D044367:Phase Transition; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D012996:Solutions; D013056:Spectrophotometry, Ultraviolet
D005296:Ferrous Compounds; D008024:Ligands; D011994:Recombinant Proteins; D012996:Solutions; D003577:Cytochrome P-450 Enzyme System; D019746:Intramolecular Oxidoreductases; C022461:prostacyclin synthetase; C034236:ferrous oxide; D010100:Oxygen