The research outlined in this proposal seeks to further our understanding of the factors that govern C-H bond activation in cytochrome P450 catalysis. Over the past several years my group has had made significant contributions to this area. Our results have impacted not only the way people think about P450 catalysis but also metal-oxo mediated C-H bond activation in general. We have led the way in the capture and characterization of critical intermediates in the P450 catalytic cycle and developed theories to describe how Nature biases enzymes for C-H bond activation. Still, much remains to be done. Our understanding of the factors that govern C-H bond activation in P450s remains incomplete. Importantly, results from our last funding period have shown that P450 can serve as a platform from which to attack some of the most important and fundamental questions in the field of C-H bond activation. There is currently a debate in the field about the factors that govern reactivity in metal-oxo driven C-H bond activation. The debate centers on whether ground state thermodynamics play the dominant role in determining reactivity or whether unpaired spin-density on the oxo ligand can provide an intrinsic lowering of the activation barrier. The examination of this fundamental issue has been hindered not only by the difficulty of measuring these quantities for reactive high-valent species but also by the lack of a series of isoelectronic and isostructural compounds over which these quantities can be varied. Our preliminary data show that P450 can fill this void. Innovations, from the last funding period, will allow us use P450 to measure the ground state thermodynamics of C-H bond activation (i.e. D(O-H), E0I, and pKaII), quantify the degree of oxyl-radical character in compound I, and, importantly, track how these quantities (and the reactivity towards C-H bonds) change as a function of electron donation from the axial ligand. The experiments outlined in this proposal will thus use an isoelectronic and isostructural system (cytochrome P450) to determine the importance of tunneling, thermodynamics, oxyl-radical character, and strong axial electron- donation in promoting C-H bond activation. There is currently no other system, synthetic or biological, that allows for a similar set of measurements and discovery. These experiments and others will evaluate our understanding of the electronic and geometric structures of compound I as well as the protective role of P450's axial thiolate ligand. We have proposed that P450's thiolate ligand can decrease the driving force for non- productive oxidations of the protein superstructure, effectively governing the partition between productive and non-productive oxidations, biasing the system towards C-H bond activation. This theory, which depends on the interplay of the one-electron reduction potential of compound I, the pKa of compound II, and the control of proton flow via substrate positioning and enzyme architecture, remains to be verified. Innovations from the last funding period will allow us to test this hypothesis.
Cytochrome P450s are responsible for the Phase I metabolism of ~75% of known pharmaceuticals. These enzymes function through the controlled activation of inert C-H bonds, a Holy Grail of synthetic chemistry. As a result of breakthroughs and innovations during the last funding period, we are in a position to answer fundamental questions about how P450s function, providing critical insights for enzyme/catalyst design.
