Selenocysteine-ligated Cytochrome P450 Intermediates

Open Access
Author:
Onderko, Elizabeth Lois
Graduate Program:
Chemistry
Degree:
Doctor of Philosophy
Document Type:
Dissertation
Date of Defense:
October 30, 2015
Committee Members:
  • Michael Thomas Green, Dissertation Advisor
  • Michael Thomas Green, Committee Chair
  • Carsten Krebs, Committee Member
  • Squire J Booker, Committee Member
  • Ming Tien, Committee Member
Keywords:
  • cytochrome P450
  • selenocysteine
  • compound I
  • compound II
  • peroxynitrite
Abstract:
Cytochrome P450s are thiolate-ligated heme enzymes that are capable of performing challenging C-H bond activation reactions. P450s are responsible for the metabolism of many pharmaceuticals as well as the biosynthesis of steroids and other biomolecules. The electron donating ability of the thiolate ligand is believed to be responsible for their ability to oxidize unactivated C-H bonds. This proposed role of the thiolate ligand was investigated using several axial ligand variants. Cysteine-containing axial ligand variants of histidine-ligated heme enzymes were successful in reproducing some spectroscopic features of P450s, but not their reactivity. Axial ligand variants of P450s where cysteine was replaced with histidine, methionine, and serine suffered from poor heme incorporation and misfolding as well as negligible levels of monooxygenase activity. Investigations of axial ligand variants where cysteine was replaced with the more electron-donating selenocysteine indicated that these variants could still perform monooxygenase reactions. However, the intermediates directly involved, compound I and compound II, were not observed. We have produced a selenocysteine-ligated variant of the P450, CYP119, and successfully trapped a selenolate-ligated compound I species. While the UV-visible spectrum resembles the WT CYP119-I spectrum, a larger quadrupole splitting value and J/D ratio result in distinct Mössbauer and EPR spectra. Analysis of the reaction with substrate suggests that SeCYP119-I may be more reactive than the compound I of the WT enzyme. The selenolate-ligated P450 compound I species was further characterized using variable temperature Mössbauer and X-ray absorption spectroscopy (XAS). The dramatic increases in both zero field splitting and exchange coupling parameters obtained from the variable temperature Mössbauer analyses are believed to result from the intrinsic properties of the ligating selenium. However, the greater increase in J relative to D suggests that the selenocysteine ligand may allow for greater spin delocalization onto the axial ligands in compound I. The features of a selenolate-ligated P450 compound II were investigated using a selenocysteine-ligated variant of CYP158A2 (SeCYP158A2 C158S). Mössbauer and EXAFS measurements confirmed that compound II of SeCYP158A2 C158S is a ferryl hydroxide. While a pH-dependent UV-visible transition was observed, Se K-edge XANES measurements indicate that at high pH the selenocysteine ligand becomes oxidized. Peroxynitrite (PN) has been controversial in its use to generate high-valent intermediates in heme proteins. The peroxynitrite-derived P450 compound I and compound II species are unusually unreactive compared to those generated using more conventional peroxides. Furthermore, Mössbauer and UV-visible measurements have suggested that P450-PN is a nitrosyl complex. We have spectroscopically characterized P450-PN and P450-NO via Mössbauer, resonance Raman and X-ray absorption spectroscopies as well as UV-visible stopped flow spectrophotometry, confirming that the reaction of peroxynitrite with thiolate-ligated enzymes produces nitrosyl complexes.