Understanding C-H Bond Activation in Heme Proteins: The Importance of the Ferryl pKa

Open Access
Author:
Yosca, Timothy Howard
Graduate Program:
Chemistry
Degree:
Doctor of Philosophy
Document Type:
Dissertation
Date of Defense:
October 05, 2012
Committee Members:
  • Michael Thomas Green, Dissertation Advisor
  • Joseph M Bollinger Jr., Committee Member
  • Squire J Booker, Committee Member
  • Ming Tien, Committee Member
Keywords:
  • cytochrome p450
  • compound II
  • ferryl pKa
Abstract:
The major focus of the Green group involves the study of C-H bond activation by heme proteins and the elucidation of the factors giving rise to this potent chemistry. Because only thiolate ligated heme enzymes such as cytochrome P450 (P450), chloroperoxidase (CPO), and aromatic peroxygenase (APO) are capable of difficult hydrocarbon oxidations, we believe that Nature has specifically chosen the thiolate ligand in order to facilitate H-atom abstraction. Current evidence suggests that the strong donating nature of the thiolate elevates the pKa of the compound II (ferryl) species, and could be the reason for the increased driving force. Conversely, it is proposed that a lower ferryl pKa in histidine and tyrosine ligated heme enzymes significantly decreases the reactivity, thus preventing C-H bond activation. In an effort to provide concrete evidence for the magnitude of the proposed driving force, we set out to quantitate the thermodynamic “pKa” parameter in histidine, thiolate, and tyrosine ligated heme systems. While it is has been shown that the thiolate ligated P450-II and CPO-II intermediates are basic in nature, there is much controversy over the protonation status of histidine ligated ferryls. X-ray crystallographic studies favor long Fe-O bond lengths (~1.85 Å), indicating protonated (FeIV-OH) moieties, while EXAFS report much shorter distances (~1.65 Å), classifying them as authentic FeIV=O intermediates. If histidine ligated ferryls are basic, then theory suggests that they could be able to activate C-H bonds, yet no experimental evidence supports this claim. In order to investigate this controversy, we explored the protonation status of the histidine ligated myoglobin compound II (Mb-II) intermediate over a wide pH range (9.5→3.9). Using a battery of spectroscopic techniques (Mössbauer, Resonance Raman, and EXAFS) our results definitively show that Mb-II is an authentic FeIV oxo with a pKa ≤ 2.65. We can infer from this study that all histidine ligated ferryls are FeIV oxos (at physiological pH), and that previous crystallographic reports for protonated ferryls in these systems are a direct result of radiation damage. Although we could only establish an upper limit for the pKa in histidine ligated proteins, we were able to directly measure the compound II pKa in both thiolate (CYP158A2 with pKa ~ 12) and tyrosine (Helicobacter pylori catalase with pKa ~ 13) ligated systems. These values represent the first ever reported pKas for any FeIV-OH species. Relative to histidine ligated enzymes, the elevated pKa in thiolate and tyrosine ligated ferryls could account for an additional ~ 13 kcals of driving force in a given hydrocarbon oxidation. Unexpectedly, tyrosine ligated hemes have higher ferryl pKas than their thiolate ligated counterparts, suggesting that CPO, APO, and P450 may not be the only heme proteins capable of C-H bond activation.