Spectroscopic characterization of the oxyferrous complex of prostacyclin synthase in solution and in trapped sol-gel...

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.