Variety in the Coupling of Mesoporphyrin IX to Apohorseradish Peroxidase C Studied by Energy Selected Fluorescence Excitation and Vibronic Hole Burning Spectroscopy

Abstract

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 (B 1 ) and two of those produced by photobleaching (B 2 , B 3 ) 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 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 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 porphyrinprotein 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 (B 1 ), in agreement with data of pressure tuning and of Stark effect hole burning studies on the same complexes.

Energy Selection Is Not Correlated in the Qx and Qy Bands of a Mg-Porphyrin Embedded in a Protein

“…In various hyperquenched glassy solvents a ϳ1-ps relaxation time from Q y to Q x was observed for Al-phthalocyanine (Reinot et al, 1999). In our previous study on vibrational relaxation in HRP the relaxation time varied in the range of 1-10 ps for different vibrational modes of pyrrole tautomers of the chromophore (Herenyi et al, 1998). We interpreted the variations based on the identification of the vibrational mode and its possible coupling to the phonon bath of the protein.…”

Dissimilar flexibility of α and β subunits of human adult hemoglobin influences the protein dynamics and its alteration induced by allosteric effectors

“…Let us choose the n 0 ′( ν ) function which is a possible representation of the so called inhomogeneous distribution function (IDF). Since the source of this broadening is the varying chromophore environment, this function is characteristic for the distribution of chromophore molecules in their environments [ 51 ].…”

Hole-Burning Spectroscopy as a Probe of Nano-environments and Processes in Biomolecules: A Review

“…This allows for monitoring the effect of external perturbations on the spectral holes because the holes are much narrower than the absorption bands and the magnitude of the external perturbation required to observe visible changes is correspondingly reduced. Hole-burning spectroscopy has been employed with a number of biologically relevant samples: heme proteins (including myoglobin, 1–10 hemoglobin, 11,12 cytochrome proteins, 13–19 peroxidase proteins 20–25 ), green fluorescent protein 26,27 and its mutants, 28–30 nucleic acids, 31–39 substructures of water, 40–49 and cellular studies. 50–52…”