The coupling between the heme and the surrounding protein in horseradish peroxidase was studied after substituting the iron protoheme by mesoporphyrin IX to produce a sample measurable by high-resolution fluorescence spectroscopy. The inner ring phototautomerization of mesoporphyrin was used to create a variety of prosthetic group configurations that were shown to be stable at cryogenic temperatures. Due to the properties of the heme crevice, some tautomeric states are characterized by distinct spectral bands. The original band of the tautomeric form stable at room temperature (B1) and two of those produced by photobleaching (B2, B3) could be selectively studied by two techniques, i.e., energy selected fluorescence excitation and vibronic hole burning spectroscopy. The line narrowed spectra were similar in the cases of complexes B2 and B3, while both are different from that of B1. From these spectra, four characteristic vibronic lines were selected and further studied by spectral hole burning experiments. The unusual shapes of some spectral holes were discussed and interpreted on the basis of a new approach to the principles of energy selected spectroscopy. Vibronic relaxation times were determined and found in the range of 1-11 ps. It could be shown that in the porphyrin-protein complexes created photochemically at low temperature, some specific vibronic modes are characterized by significantly increased relaxation time values. It was thus experimentally verified that the coupling to the protein is the strongest in the lowest energy configuration stable at room temperature (B1), in agreement with data of pressure tuning and of Stark effect hole burning studies on the same complexes.
ASJC Scopus subject areas
- Physical and Theoretical Chemistry
- Surfaces, Coatings and Films
- Materials Chemistry