Structural, electronic, and theoretical characterization of compound i in P450s, with a focus on x-ray absorption spectroscopy

Open Access
Krest, Courtney Marie
Graduate Program:
Doctor of Philosophy
Document Type:
Date of Defense:
August 24, 2012
Committee Members:
  • Michael Thomas Green, Dissertation Advisor/Co-Advisor
  • Michael Thomas Green, Committee Chair/Co-Chair
  • Joseph M Bollinger Jr., Committee Member
  • Carsten Krebs, Committee Member
  • Ming Tien, Committee Member
  • Cytochrome P450
  • Compound I
  • Purification
  • XAS
  • Metal K-edge
  • Ligand K-edge
  • Mössbauer
  • Exchange coupling
C-H bonds are ubiquitous and inert in Nature. Much research stems from the desire to activate these unfunctional groups to more reactive entities. The heme-containing enzymes, cytochrome P450s (P450s) can do this chemistry with ease. P450s are unique thiolate ligated heme enzymes responsible for detoxification in many organisms. Recently, the active oxidant responsible for C-H bond activation by P450s, was trapped in high yield by the Green Lab. This thesis delves into the structural and electronic characterization of this species and its comparison to a similar intermediate in the thiolate ligated heme peroxidase, chloroperoxidase. To gain knowledge of this reactive intermediate we turned to Fe and sulfur K-edge X-ray absorption spectroscopy, variable temperature Mössbauer spectroscopy, and density functional theory calculations. Abstracts of each chapter are below. Chapter 2 P450s are of interest due to their ability to activate ubiquitous and inert C-H bonds. Only recently has the active intermediate responsible for this reaction been trapped and characterized in the P450 CYP119. Herein, we report the capture and spectroscopic characterization of a second high yield preparation of compound I in another P450, P450ST, in ~ 65% yield. We report that P450ST-I strongly resembles CYP119-I and CPO-I via UV/Visible stopped-flow and Mössbauer spectroscopies. Electron paramagnetic resonance studies of P450ST-I reveal a similar |J|/D (ratio of exchange coupling, J, over zero field splitting, D) as found in CYP119-I, both are elevated in comparison to the |J|/D found in CPO-I. Chapter 3 Previous to the recent capture and characterization of P450 compound I (P450-I) in CYP119, most of our knowledge of the reactive intermediates in the P450 cycle was obtained from the study of chloroperoxidase (CPO). Despite similar thiolate ligated heme active sites and the ability to perform P450 like chemistry, CPO is less reactive than P450. The preparation of P450-I in high yield has afforded the opportunity for direct spectroscopic comparison of P450-I and CPO-I. Rittle and Green determined that the |J|/D of CYP119-I was greater than that of CPO-I (1.30 versus 1.02). The exact reasons for this difference were unknown but it was hypothesized that an increased value of |J| could be caused by a greater spin density on the sulfur and/or a shorter Fe-S bond. Using variable temperature Mössbauer spectroscopy, we have observed that DCYP119-I is ~1.6DCPO-I and |J|CYP119-I is ~2|J|CPO-I. To determine the factors that contribute to a greater value of |J|, we performed Fe K-edge extended x-ray absorption fine structure (EXAFS) spectroscopy on both compound I species. Analysis of EXAFS data revealed that the Fe-S distance in CYP119-I is 0.09 Å shorter than that in CPO-I. DFT studies paired with analysis of Fe XANES also reveal a shorter Fe-S bond, which corresponds to a decrease in area of the Fe pre-edge feature. A shorter Fe-S distance in CYP119-I could account for the increased reactivity of CYP119-I over CPO-I. Chapter 4 Compound I in thiolate ligated heme systems is best described as an Fe(IV) oxo anitiferromagnetically coupled to a radical that is delocalized over the porphyrin and sulfur ligands. The amount of spin density on the sulfur is an important factor that contributes to |J|, the exchange coupling of the Fe(IV) oxo moiety and the ligand radical. Axial ligand spin density may also contribute to the reactive nature of P450s. It has been difficult to pin down the exact amount of radical character on the sulfur due to the previously low yield of P450-I. Adding to the confusion were difficult techniques for radical quantification (advanced EPR techniques requiring expensive isotopes and ligand XAS) as well as unusual and conflicting theoretical predictions of sulfur spin density. Herein, we have performed sulfur K-edge XANES to gain insight into the amount of sulfur radical in CYP119-I. In accordance with the literature, a peak at 2468.4 eV, indicative of a sulfur-based radical, was seen. Upon fitting this peak we have determined that there is 10-13% radical character on the sulfur in CYP119-I and less in CPO-I. DFT calculations have been utilized to gain insight into these measurements. Chapter 5 Recently Yosca et al determined the ferryl pKa in compound II of CYP158A2 (CYP158A2-II) using Mössbauer and stopped-flow UV/Visible absorption spectroscopies of samples of varying pH. As part of his research he characterized both the high pH (unprotonated) and low pH (protonated) forms of compound II via EXAFS spectroscopy. Briefly discussed were the Fe K-edge XAS data including the vast difference in pre-edge intensity between the two forms of compound II. Herein, we have analyzed the Fe K-edge XANES as well as the S K-edge XANES of CYP158A2 ferric, unprotonated compound II, and protonated compound II via experiment and computation. Calculations could reproduce experimental pre-edge features relatively well.