More than two pyrrole tautomers of mesoporphyrin stabilized by a protein. High resolution optical spectroscopic study

Abstract

Mesoporphyrin IX substituted horseradish peroxidase was studied by fluorescence line narrowing and hole burning techniques at cryogenic temperatures. The spectral data reveal that four pyrrole tautomeric configurations of the chromophore are populated within the protein under the influence of irradiation and/or thermal treatment, and the existence of a fifth and a sixth tautomeric configuration is also likely. The relative population of the tautomers changes upon deuterium substitution through modification of the phototransition rate, and also depends on pH, which changes the protonation of neighboring amino acids in the heme pocket. The energy separation of the origins of the tautomers is approximately 100 cm-1. The distribution of barrier heights separating the different tautomeric forms in the ground state and their distribution was determined by temperature cycling hole burning. The distributions can be approximated by Gaussians. The experiment directly yields the distributions on a relative temperature scale, and a model is presented to transform the barrier heights into energy values. It is suggested that the energies for the tautomers are split partially due to the protein crystal field and that the trapping of the tautomeric forms is the consequence of interactions with neighboring amino acids within the heme pocket.

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

“…where A 0 and A T are the hole areas at the beginning and after the cycling to a temperature T, R 0 is the pre-exponential rate factor in the kinetics of the activated process, and k is the Boltzmann constant (Kohler et al, 1988;Fidy et al, 1992). After hole burning at 5 K, the sample was warmed up to a certain cycling temperature and kept there for 5 min; then it was cooled back to 5 K where the spectrum was recorded.…”

Protein dynamics at low temperatures

“…The hole burning reaction is a light induced transfer of the inner ring protons of the chromophore mesoporphyrin-IX. 38 Burning times and powers were of the order of 16 s and 1 W, respectively. Reading of the holes was performed in the transmission mode.…”

Protonation of porphyrin in iron-free cytochromec: Spectral properties of monocation free base porphyrin, a charge analogue of ferric heme

“…For native Fe(II) cyt c, a splitting in the Q 0,0 absorption band is interpreted to indicate that the asymmetric protein environment reduces the symmetry of the heme 37 and in metal-free porphyrin proteins differences in the absorption and emission properties of different tautomers can be detected by high resolution techniques. 38 High resolution techniques would be required to see whether the different arrangements of pH ϩ can be detected, and whether these account for observed spectral differences between folded and unfolded spectra.…”