VARIETY IN THE COUPLING OF MESOPORPHYRIN-IX TO APOHORSERADISH PEROXIDASE-C STUDIED BY ENERGY-SELECTED FLUORESCENCE EXCITATION AND VIBRONIC HOLE-BURNING SPECTROSCOPY

The coupling between the heme and the surrounding protein in horseradi sh peroxidase was studied after substituting the iron protoheme by mes oporphyrin IX to produce a sample measurable by high-resolution fluore scence spectroscopy. The inner ring phototautomerization of mesoporphy rin was used to create a variety of prosthetic group configurations th at were shown to be stable at cryogenic temperatures. Due to the prope rties of the heme crevice, some tautomeric states are characterized by distinct spectral bands. The original band of the tautomeric form sta ble at room temperature (B-1) and two of those produced by photobleach ing (B-2, B-3) could be selectively studied by two techniques, i.e., e nergy selected fluorescence excitation and vibronic hole burning spect roscopy. The line narrowed spectra were similar in the cases of comple xes B-2 and B-3, while both are different from that of B-1, From these spectra, four characteristic vibronic lines were selected and further studied by spectral hole burning experiments. The unusual shapes of s ome spectral holes were discussed and interpreted on the basis of a ne w approach to the principles of energy selected spectroscopy. Vibronic relaxation times were determined and found in the range of 1-11 ps. I t could be shown that in the porphyrin-protein complexes created photo chemically at low temperature, some specific vibronic modes are charac terized by significantly increased relaxation time values. It was thus experimentally verified that the coupling to the protein is the stron gest in the lowest energy configuration stable at room temperature (B- 1), in agreement with data of pressure tuning and of Stark effect hole burning studies on the same complexes.