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

Abstract

The Qx-Qy splitting observed in the fluorescence excitation spectra of Mg-mesoporphyrin-IX substituted horseradish peroxidase (MgMP-HRP) and of its complex with naphthohydroxamic acid (NHA) was studied by spectral hole burning techniques. The width of a hole directly burnt in the Qy band and that of a satellite hole indirectly produced in Qy as a result of hole burning in Qx was compared. We also studied the dependence of the satellite hole in the Qy band on the burning frequency used in the Qx band. Both the directly and indirectly burnt holes were very broad in the (higher energy) Qy band. The width of the satellite hole in the Qy band was equal to the entire width of the inhomogeneously broadened band, independently from the position of hole burning in Qx. This is indicative of a clear lack of correlation between the electronic transition energies of the Qx and Qy bands. A photoproduct was produced by laser irradiation of the MgMP-HRP/NHA complex and was identified as a species with lowered Q-splitting. Conversion of the photoproduct could be achieved by thermal activation measured in temperature-cycling experiments, with a characteristic temperature of 25 K. We attribute the phototransformation to a conformational change of MgMP.

Electronic and Vibronic Contributions to the Band Splitting in Optical Spectra of Heme Proteins

“…We like to emphasize that our study is not aimed at analyzing the various Q-band splits reported in the literature. [23][24][25][28][29][30][31] The results of our study rather suggest that the Q-band spectrum alone does not provide a sufficient basis for such an analysis, because the observed splits and intensity redistributions can be accounted for by various combinations of electronic and vibronic perturbations. In principle, the latter can only be determined from the resonance excitation profiles and depolarization ratio dispersion of at least all those modes, which display significant Raman intensity.…”

“…24 It should be emphasized that cytochrome c is not the only heme protein that exhibits a splitting of the optical bands. A series of optical absorption and fluorescence studies by Fidy and co-workers have revealed significant Q-band splitting for Mg-meso-porphyrins incorporated into the matrix of the isoenzyme C of horseradish peroxidase, 28 which were found to increase upon binding of naphthohydroxamic acid. 29 Ko ¨hler et al 30 obtained similar results by combining Stark spectroscopy and spectral hole burning.…”

Measuring the Ultrafast Correlation Dynamics between the Qxand QyBands in Chlorophyll Molecules

“…Friedrich et al reported the frequency correlation of the Q bands in Mg-mesoporphyrin horseradish peroxidase (MgMP-HRP) using spectral hole burning spectroscopy. 6 Their results indicate a lack of correlation between the two bands. This is based on a broad non-resonant satellite hole in the Qy band observed when a selective subpopulation of the Qx band in an inhomogeneously broadened spectra was burnt.…”

“…There have not been many studies on the correlation between the Q x and Q y transitions. Fidy et al reported the frequency correlation of the Q bands in Mg-mesoporphyrin horseradish peroxidase using spectral hole burning spectroscopy . Their results indicate a lack of correlation between the two bands.…”

Structural Disorder of Native Horseradish Peroxidase C Probed by Resonance Raman and Low-Temperature Optical Absorption Spectroscopy

“…Shelnutt, private communication). With respect to HRP, fluorescence and absorption measurements have revealed a splitting of the Q band at low temperatures for HRP−CO, HRP−CO complexed with benzohydroxamic acid (BHA), , and Mg-mesoporphyrin-IX substituted HRP. , However, a detailed analysis and characterization of the distortions giving rise to the observed band splitting is outstanding.…”