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

The electronic structure of the heme oxy-iron center in oxyhemoglobin and oxymyoglobin has been the subject of debate for decades. Various experimental and computational methods have been used to study this system, leading to conflicting conclusions. This study uses X-ray spectroscopy to directly probe the iron center in the highly delocalized oxyhemoglobin and its model compound to define the electronic structure and understand the differences between the protein and the model. This study settles a longstanding debate in bioinorganic chemistry and provides insight into heme iron–oxygen binding, the key first step in many biocatalytic processes.Hemoglobin and myoglobin are oxygen-binding proteins with S = 0 heme FeO28 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 FeO28 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 FeII and FeIII [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 FeII, while oxyhemoglobin is qualitatively similar to FeIII, 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 FeII with 6–8% FeIII character, while oxyhemoglobin has a very mixed wave function that has 50–77% FeIII character and a partially polarized Fe–O2 π-bond.