Spin states of myoglobin, which is one of hemoproteins, in aqueous solutions at room temperature were probed from the porphyrin C=N π* peaks of heme using nitrogen K-edge X-ray absorption spectroscopy combined with inner-shell quantum chemical calculations. Although the protein polypeptide chains include numerous nitrogen atoms, the porphyrin C=N π* peaks were analyzed to distinguish them from the protein polypeptide peaks. Oxymyoglobin with an Fe 2+ ion connected to an oxygen molecule is in the S = 0 state. Deoxymyoglobin with an Fe 2+ ion shows a spin equilibrium between the S = 2 and 1 states. Metmyoglobin with an Fe 3+ ion connected to a water molecule also shows a spin equilibrium between the S = 5/2 and 3/2 states. These results clearly propose that the spin states of hemoproteins should be investigated in physiological conditions because these are influenced by factors such as liquid temperature, solvent, gas adsorption, and protein structure.
Myoglobin is a hemoprotein that stores oxygen molecules in muscle tissue and is composed of a heme and a polypeptide chain. As shown in Fig. 1, deoxymyoglobin (deoxyMb) contains a five-coordinated heme with a Fe 2+ ion. Oxymyoglobin (oxyMb) contains a six-coordinated heme with a Fe 2+ ion connected to an oxygen molecule. Metmyoglobin (metMb) contains a six-coordinated heme with a Fe 3+ ion connected to a water molecule. Despite many studies, the spin states of myoglobin have not yet been understood because they are influenced by the interactions with protein polypeptide chains. It is necessary to investigate the spin states of myoglobin in physiological conditions. Herein, the spin states of different forms of myoglobin heme iron in aqueous solution at room temperature were investigated using the N K-edge X-ray absorption spectroscopy (XAS) combined with inner-shell quantum chemical calculations.
The XAS measurements were performed at the soft X-ray beamline BL3U of the UVSOR-III synchrotron facility using a transmission-type liquid cell, in which the liquid layer was sandwiched between two 100 nm-thick SiC membranes and the thickness of the liquid layer was precisely controlled from 20 nm to 40 μm. Figure 1 shows the N K-edge XAS spectra of oxyMb, deoxyMb, and metMb at a concentration of 1 mM in aqueous solutions at 25 °C. For obtaining appropriate absorbance of soft X-rays, the thickness of aqueous oxyMb solution was set to be 18 μm. The protein polypeptide chains contain numerous nitrogen atoms and show a strong peak at 401.4 eV. Because the porphyrin C=N π* peaks exist approximately at 400.0 eV, the porphyrin C=N π* peaks can be distinguished from the protein polypeptide peaks.
The spin states of myoglobin heme irons were analyzed from the porphyrin C=N π* peaks compared with the inner-shell calculations. The XAS spectrum of oxyMb exhibits a single C=N π* peak and exclusively shows the S = 0 state. By contrast, the XAS spectra of deoxyMb and metMb exhibit two C=N π* peaks. DeoxyMb shows a spin equilibrium between the S = 2 and 1 states. Because of the larger intensity of the first C=N π* peak compared to the second peak, deoxyMb in the S = 2 state is favored over the S = 1 state. The S = 0 state of oxyMb results from antiferromagnetism with the oxygen molecule in the S = 1 state. MetMb shows a spin equilibrium between the S = 5/2 and 3/2 states. Because the intensity of the second C=N π* peak is larger than that of the first peak, metMb in the S = 3/2 state is favored over the S = 5/2 state. Because the porphyrin C=N π* peaks reflect the electronic structures and spin multiplicities of heme due to metal‒ligand delocalization, the N K-edge XAS is suitable to investigate the spin equilibriums of hemoproteins in the physiological conditions, which are influenced by factors such as liquid temperature, solvent, gas adsorption, and protein structure.
Physical Chemistry Chemical Physics
Experimental study
Not applicable
Spin states of myoglobin heme iron in aqueous solutions at room temperature probed from porphyrins using nitrogen K-edge X-ray absorption spectroscopy
24-Aug-2026