Iron(IV)hydroxide pKa and the Role of Thiolate Ligation in C-H Bond Activation by Cytochrome P450

Saturday, May 31, 2014

Cytochrome P450s (P450s) are a family of monooxygenase enzymes that are nearly ubiquitous in nature. P450s are often described as biological blowtorches due to their incredible oxidizing power:1 They can hydroxylate C-H bonds of about 98-100 kcal/mol.  P450s are responsible for the phase I metabolism of 75% of all pharmaceuticals in vivo and are thusly referred to as nature’s detoxifier.2 This study focuses on how a protein can perform such difficult, oxidizing reactions without damaging its own fragile protein superstructure. A full understanding of the factors that govern P450 chemistry have wide implications not only for the medical and biological fields but also in synthetic applications.

Briefly, the generally accepted mechanism for C-H bond activation by P450s starts with the ferric enzyme binding a substrate and converting to a high spin species.  Next, the enzyme is reduced to its ferrous state in which dioxygen binds, forming a species best described as a ferric superoxide complex. This step is followed by the reduction to a ferric peroxo species. This species, in turn, is protonated to form a ferric hydroperoxo complex. Addition of another proton cleaves the O-O bond, forming compound I - an iron(IV) oxo species with a ligand-based radical - and water.  Compound I then abstracts hydrogen from the substrate to yield compound II - an iron(IV) hydroxide species - and a substrate radical which recombine to form the hydroxylated product and ferric enzyme.3

One hypothesis of how P450s can perform such difficult reactions without damaging their fragile protein superstructures is their unique thiolate ligation.  The thought is that this axial thiolate ligand (as compared to histidine-ligated heme systems) allows for an increase in the pKa of compound II (an Fe(IV)-OH called the “rebound intermediate”), which correspondingly lowers the reduction potential of compound I (an Fe(IV)=O, the active intermediate in P450 chemistry). This results in a decreased driving force for auto-oxidation (destruction) of fragile residues in the delicate protein.

Through a combination of rapid freeze quench and a variety of spectroscopies including Mössbauer, stopped flow UV/Vis, and x-ray absorption (Beam Line 7-3 at SSRL), the researchers were able to pinpoint the pKa of the ferryl species in compound II to be 11.9, which is elevated in comparison to the pKa of compound II in histidine-ligated heme systems that cannot perform the same difficult reactions as P450s.

Figure 1

Mössbauer spectroscopy showed the formation of a new species at elevated pH.  The parameters of this species (narrower quadrupole splitting) were consistent with the formation of an authentic Fe(IV) oxo species.  Stopped flow UV/Vis spectroscopy also showed reversible spectral changes with multiple isosbestic points indicating that these pH-driven changes were resulting from deprotonation of the hydroxo species with increasing pH. Data from Mössbauer and stopped-flow UV/Vis experiments were used to construct pH titration curves that indicate a pKa of 11.9, figure 1.  Using Marcus theory, it was determined that this elevated pKa results in a greater than 10,000 fold reduction in the rate constant of nonproductive oxidations (i.e. of protein residues) versus histidine-ligated systems.  This eliminates these autoxidation reactions as competition for substrate reactions. It was concluded that these results could explain why there are no histidine-ligated P450s.

Figure 2

XAS was used to confirm that the low pH and high pH species were in fact the hydroxide and oxo forms, respectively. The results are shown in figure 2.  Both high and low pH species had Fe K-edges that lie 1.5 eV above the ferric edge, which is consistent with an Fe(IV) species.  However, the low pH species has an Fe-O bond distance of 1.84 Å indicative of an Fe(IV)-OH species while the high pH species had and Fe-O bond of 1.68 Å indicative of an Fe(IV)=O species.

This work was supported by NIH (R01-GM101390).  Portions of this research were carried out at the Stanford Synchrotron Radiation Lightsource (SSRL), a Directorate of SLAC National Accelerator Laboratory and an Office of Science User Facility operated for the U.S. Department of Energy (DOE) Office of Science by Stanford University. The SSRL Structural Molecular Biology Program is supported by the DOE Office of Biological and Environmental Research and by the NIH National Institute of General Medical Sciences (including P41GM103393).

  1. I. Schlichting, J. Berendzen, K. Chu, A. Stock, S. Maves, D. Benson, R. Sweet, D. Ringe, G. Petsko and S. Sligar, Science 287, 1615 (2000)
  2. J. Rittle and M. Green, Science 330, 933 (2010)
  3. C. Krest, E. Onderko, T. Yosca, J. Calixto, R. Karp, J. Livada, J. Rittle, and M. Green, J. Biol. Chem. 88, 17074 (2013)
Primary Citation: 

T. H. Yosca, J. Rittle, C. M. Krest, E. L. Onderko, A. Silakov, J. C. Calixto, R. K. Behan and M. T. Green, "Iron(IV)hydroxide pKa and the Role of Thiolate Ligation in C-H Bond Activation by Cytochrome P450", Science 342, 825 (2013), DOI: 10.1126/science.1244373

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