Much work has been performed to study reactive electron orbitals in nonheme enzymes (NHE) and their activation of oxygen. Similar to NHEs, heme-containing enzymes play a diverse set of roles in the biosphere and factor heavily to human health: detoxification, oxygen transport, and hormone synthesis. It is important that heme enzymes be understood to a similar extent as NHEs, however the tools used to characterize NHE electron covalency of Fe, such as magnetic circular dichroism optical spectroscopy, are not as applicable to hemes due to the highly delocalized equatorial porphyrin ring that obscures the covalency of the Fe atom. The covalency of the low-lying valence orbitals of the heme site, actively tunes intermediates for their function in biology. To directly probe the Fe center in heme enzymes, this project will use the relatively new resonant inelastic X-ray scattering (RIXS) spectroscopy. 1s2p RIXS uses K-edge, hard X-ray, incident photons and detects the subsequent 2p to 1s hole-filling resulting in the same final state as L-edge X-ray absorption spectroscopy (XAS) ? with different selection rules. RIXS yields information about the electronic structure of the frontier molecular orbitals (FMOs), specifically d orbital covalency, critical to studying oxygen activation by hemes, however without the weaknesses inherent in soft X-ray L-edge XAS such as high vacuum requirements, high sample concentrations, and fluorescence inhibition. However, a new ultra-low noise detector called the TES will finally allow Fe L-edge XAS spectra of dilute enzyme samples, and correlating 1s2p RIXS with L-edge XAS will afford the differential orbital covalency of the frontier molecular orbitals that are key to reactivity. Initially, the trainee will apply RIXS and L- edge XAS to nonheme and heme model complexes, particularly the well-understood nonheme models will allow development of the experimentally observed 4p orbital mixing into the simulation of RIXS spectra. The trainee will then explore the change of the Fe=O bond when going from a nonheme to a heme environment in an enzyme. With L-edge XAS and RIXS the reactive orbitals of cytochrome p450 compound II will reveal the driving forces for the ?rebound mechanism? of hydroxylation. Next, this study will explore the change in the Fe=O bond upon conversion to the compound I radical cation intermediate and the implications for H-atom abstraction. Finally, the frontier molecular orbitals of compound I will be compared across the different trans axial ligations in heme enzymes. This interchange of the axial ligand (histidine, cysteine, and tyrosine) will quantitively identify the, currently, loosely defined ?push? and ?pull? effects on the heme electronic structure that allow heterolytic O2 cleavage. This project will further the fundamental knowledge of Fe oxygen chemistry in heme, and nonheme, enzymes. Providing new insights into the nature and reactivity of the Fe=O bond, the role that the porphyrin plays in hemes, and how that is adapted in NHEs where no such electron sink is available. This work will also develop the methods and modelling of 1s2p RIXS and TES detected L-edge XAS on bioinorganic systems.
Heme-containing enzymes play a crucial role in human health, composing the largest gene superfamily in the human genome, with roles in: oxygen transport, steroid biosynthesis and metabolism, cholesterol biosynthesis, and drug metabolism. Their diverse chemistry relies on activation of O2 by heme, a cofactor that contains iron surrounded by a porphyrin macrocycle that completely obfuscates the central iron atom from standard optical spectroscopic methods. This project uses an innovative spectroscopy called resonant inelastic X-ray scattering and develops a new type of detection for L-edge X-ray absorbance spectroscopy to make unprecedented observations of the activation of O2 by the iron in heme-containing enzymes to better understand the chemistry of heme enzymes related to the chemistry of their non-heme containing counterparts with respect to their impact on human health.
