Open Access
Behan, Rachel Koren
Graduate Program:
Doctor of Philosophy
Document Type:
Date of Defense:
December 07, 2007
Committee Members:
  • Michael Thomas Green, Committee Chair
  • Joseph M Bollinger Jr., Committee Member
  • Carsten Krebs, Committee Member
  • Patrick Cirino, Committee Member
  • ferryl
  • heme
  • cytochrome P450
  • peroxynitrite
Our group is particularly interested in the oxygen activation chemistry performed by heme peroxidases and cytochrome P450s. We set out to study compound II in cytochrome P450 and peroxidases because the identity of this intermediate is still the subject of debate. Spectroscopic studies on compound II in peroxidases have indicated that these intermediates are Fe(IV)oxo species. X-ray crystal structures, however, have indicated long Fe-O bonds for the ferryl intermediates in horseradish peroxidase, cytochrome c peroxidase, catalase, and myoglobin suggesting that these intermediates are protonated. To verify the X-ray crystal structure results, we performed both Mössbauer and resonance Raman spectroscopies on the same sample of ferryl myoglobin. Our experiments clearly supported the existence of only the Fe(IV)oxo species from pH 3.5 to 8.5. This result implies that the pKa of ferryl myoglobin is less than 3.5 and suggests that the long Fe-O bonds from the crystal structures are a consequence of photoreduction and does not represent the true nature of the intermediate. Although ferryl myoglobin was not protonated over a wide pH range, we have shown that compound II in cytochrome P450s is protonated at physiological pH. The ferryl intermediates in P450BM3, P450cam, and CYP158 were studied by Mössbauer spectroscopy in conjunction with DFT calculations. As was observed in chloroperoxidase compound II, a large quadrupole splitting (and an Fe(IV)-like isomer shift) was obtained for the three intermediates. The experimental Mössbauer parameters also agreed well with the theoretically determined parameters for protonated ferryl models. High-field Mössbauer measurements on P450BM3 compound II also confirmed this assignment because this intermediate had an S = 1 spin state. Based on previous experiments concerning the reaction of heme enzymes with peroxynitrite, it was evident that peroxynitrite could be used as an oxidant to generate ferryl intermediates. In our quest to study the electronic structure of P450 compound II, we utilized peroxynitrite as an oxidant to generate P450BM3 compound II. On characterization of this intermediate using UV/visible, resonance Raman, and Mössbauer spectroscopies, as well as DFT calculations, we determined that the PN intermediate in P450BM3 was not a ferryl intermediate, but rather an S = 0, ferric nitrosyl species. We studied the reaction of peroxynitrite with five other thiolate-ligated heme proteins and found similar results; however, when the reaction was performed with horseradish peroxidase and cytochrome c peroxidase (histidine-ligated heme proteins) a ferryl intermediate was found.