Spin states of myoglobin in aqueous solutions at room temperature were probed from the porphyrin C=N π* peaks of heme using N K-edge XAS with inner-shell calculations. The porphyrin C=N π* peaks were separately observed from the protein polypeptide peaks that include numerous nitrogen atoms. Oxymyoglobin with an Fe2+ ion connected to an oxygen molecule is in the S = 0 state. Deoxymyoglobin with an Fe2+ ion shows a spin equilibrium between the S = 2 and 1 states. Metmyoglobin with an Fe3+ ion connected to a water molecule also shows a spin equilibrium between the S = 5/2 and 3/2 states. This study proposes that the N K-edge XAS measurements of porphyrins are effective for determining the spin equilibriums of hemoproteins, which are influenced by factors such as liquid temperature, solvent, gas adsorption, and protein structure.
