The Photoexcited Triplet State as a Probe of Chromophore-Protein Interaction in Myoglobin

Abstract

The photoexcited metastable triplet state of Mg 2+-mesoporphyrin IX (MgMPIX) or Mg 2+-protoporphyrin IX (MgPPIX) located in the heme pocket of horse myoglobin (Mb) was investigated by optical and electron paramagnetic resonance (EPR) spectroscopy, and its properties were compared with the model complexes, MgMPIX, MgPPIX, and Mg 2+ etioporphyrin I (MgETIOI), in noncoordinating and coordinating organic glasses. Zero-field splitting parameters, line shape, and Jahn-Teller distortion in the temperature range of 3.8–110 K are discussed in terms of porphyrin-protein interactions. The triplet line shapes for MgMPIXMb and MgPPIXMb show no temperature-dependent spectral line shape changes suggestive of Jahn-Teller dynamics, and it is concluded that the energy splitting is ≫150 cm −1, suggesting symmetry breaking from the anisotropy of internal electric fields of the protein, and consistent with previous predictions (Geissinger et al. 1995. J. Phys. Chem. 99:16527–16529). Both MgMPIXMb and MgPPIXMb demonstrate electron spin polarization at low temperature, and from the polarization pattern it can be concluded that intersystem crossing occurs predominantly into in-plane spin sublevels of the triplet state. The splitting in the Q 0,0 absorption band and the temperature dependence and splitting of the photoexcited triplet state of myoglobin in which the iron was replaced by Mg 2+ are interpreted in terms of effects produced by electric field asymmetry in the heme pocket.