OPTICAL FINE-STRUCTURE INVESTIGATION OF PORPHYRIN PROTEIN INTERACTIONS - MAGNESIUM AND METAL-FREE MYOGLOBIN

Fluorescence line-narrowing spectroscopy was used to evaluate the true 0,0 band shape from the population distribution of the 0,0 spectral band and to determine the excited-state vibrational spectrum of the porphyrin in Mg protoporphyrin substituted myoglobin (Mg-Mb) and protoporphyrin substituted myoglobin (HH-Mb). The population distribution function for Mg-Mb showed a bimodal distribution, with approximately equal contributions for the two components. The 0,0 energies were centered at 16 716 and 16 873 cm-1 and the widths were 106 and 66 cm-1 for the lower and higher energy forms, respectively. The population distribution for HH-Mb also showed two main components, but the contribution of the second component at lower frequency was less than for the predominant form centered at 16 127 cm-1 with width of 72 cm-1. The bimodal distribution is discussed in terms of possible ligational effects or tautomeric equilibrium. Evaluation of the energy-selected excitation spectra for Mg-Mb and HH-Mb yields the vibrational frequencies of the excited singlet state porphyrin in the range 100-1550 cm-1. Within the accuracy of the measurement, the vibrational frequencies for Mg-Mb were constant within the inhomogeneous distribution, indicating that changes in the electric field in different protein substates, rather than torsional differences in the porphyrin, lead to the inhomogeneity in the spectrum. The excited-state vibrations show a 2-20-cm-1 downshift relative to the ground-state levels and comparison with resonance Raman spectra allows tentative assignments to be made.