# Electronic Structure of Heme Enzyme Intermediates from Resonant Inelastic X-ray Scattering and L-Edge X-ray Absorption Spectroscopy

> **NIH NIH F32** · STANFORD UNIVERSITY · 2020 · $47,760

## Abstract

Project Summary/Abstract
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 ...

## Key facts

- **NIH application ID:** 10241897
- **Project number:** 5F32GM122194-04
- **Recipient organization:** STANFORD UNIVERSITY
- **Principal Investigator:** LELAND BRUCE GEE
- **Activity code:** F32 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2020
- **Award amount:** $47,760
- **Award type:** 5
- **Project period:** 2017-09-16 → 2021-04-30

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10241897

## Citation

> US National Institutes of Health, RePORTER application 10241897, Electronic Structure of Heme Enzyme Intermediates from Resonant Inelastic X-ray Scattering and L-Edge X-ray Absorption Spectroscopy (5F32GM122194-04). Retrieved via AI Analytics 2026-05-21 from https://api.ai-analytics.org/grant/nih/10241897. Licensed CC0.

---

*[NIH grants dataset](/datasets/nih-grants) · CC0 1.0*