Polarization anisotropy identifies bound azide in photoexcited ferric myoglobin: Resolving photodeligation ambiguity by femtosecond infrared spectroscopy

Abstract Polarization‐resolved femtosecond time‐resolved infrared spectroscopy was employed to elucidate the origin of a transient absorption band in azide‐bound ferric myoglobin (MbN 3 ) following Q‐band excitation. Previous studies attributed this feature to a low‐spin to high‐spin transition during electronic relaxation of the photoexcited MbN 3 , but ambiguity remained regarding possible photodissociation. By analyzing polarization anisotropy, we directly determined the orientation of the azide ligand associated with the transient signal. The anisotropy of the new absorption band is identical to that of the bound state, yielding an angle of 53° ± 1° relative to the heme normal, consistent with a bound configuration. This result rules out photodissociation, which would produce markedly different anisotropy. The transient spectral evolution is therefore attributed to a spin‐state transition within the bound state following photoexcitation. These findings demonstrate that MbN 3 does not undergo photodeligation under visible Q‐band excitation and provide clear mechanistic insight into the photophysics of anion‐bound ferric heme proteins.