Investigations of Heme Protein Absorption Line Shapes, Vibrational Relaxation, and Resonance Raman Scattering on Ultrafast Time Scales

Abstract

The spectral dynamics of photoexcited myoglobin (Mb) and its ligated species have been measured using both single wavelength and broadband continuum probes. Ultrafast spectral relaxation of the deoxy photoproduct involves an initially broadened and red-shifted absorption band, which is observed for all samples studied (deoxyMb, metMb, MbNO, MbCO). These results are consistent with an immediate (sub 100 fs) relaxation to the electronic ground state followed by vibrational equilibration. Relaxation of the “hot” photoproduct spectrum is well described using independent time scales for narrowing (400 fs) and blue shifting (0.4−4 ps) as the system returns to equilibrium. A previous multiple electronic intermediate state model, which is based on single wavelength measurements (Petrich, J. W.; Poyart, C.; Martin, J. L. Biochemistry 1988, 27, 4049), does not adequately explain the observed broadband spectral dynamics, particularly on the blue side of the Soret band (unless still more electronic states are postulated). The vibrational relaxation pathway in Mb is explored by using samples with a modified local heme environment (e.g., His93 → Gly mutation and protoporphyrin IX → porphine substitution). The His93 → Gly mutation experiment demonstrates that the covalent bond between the iron and the proximal histidine has little effect on the overall vibrational relaxation of the “hot” heme. In contrast, the protoporphyrin IX → porphine substitution experiment demonstrates the importance of the van der Waals contacts between the heme and the protein/solvent matrix in cooling the locally hot heme. Finally, we discuss the effects of the observed broadband spectral dynamics on time-resolved resonance Raman intensities and show how the time dependent line shape function plays an important role in the extraction of mode specific vibrational temperatures from the resonance Raman data.