Resonant inelastic X-ray scattering determination of the electronic structure of oxyhemoglobin and its model complex

Hemoglobin and myoglobin are oxygen-binding proteins with S = 0 heme {FeO₂}⁸ active sites. The electronic structure of these sites has been the subject of much debate. This study utilizes Fe K-edge X-ray absorption spectroscopy (XAS) and 1s2p resonant inelastic X-ray scattering (RIXS) to study oxyhemoglobin and a related heme {FeO₂}⁸ model compound, [(pfp)Fe(1-MeIm)(O₂)] (pfp = meso-tetra(α,α,α,α-o-pivalamido-phenyl)porphyrin, or TpivPP, 1-MeIm = 1-methylimidazole) (pfpO₂), which was previously analyzed using L-edge XAS. The K-edge XAS and RIXS data of pfpO₂ and oxyhemoglobin are compared with the data for low-spin Fe^II and Fe^III [Fe(tpp)(Im)₂]^0/+ (tpp = tetra-phenyl porphyrin) compounds, which serve as heme references. The X-ray data show that pfpO₂ is similar to Fe^II, while oxyhemoglobin is qualitatively similar to Fe^III, but with significant quantitative differences. Density-functional theory (DFT) calculations show that the difference between pfpO₂ and oxyhemoglobin is due to a distal histidine H bond to O₂ and the less hydrophobic environment in the protein, which lead to more backbonding into the O₂ A valence bond configuration interaction multiplet model is used to analyze the RIXS data and show that pfpO₂ is dominantly Fe^II with 6-8% Fe^III character, while oxyhemoglobin has a very mixed wave function that has 50-77% Fe^III character and a partially polarized Fe-O₂ π-bond.