The protoporphyrin-apoperoxidase complex as a horseradish peroxidase analog: A fluorimetric study of the heme pocket

Abstract

Similarity of the protein tertiary structures of the native horseradish peroxidase (donor: hydrogen-peroxide oxidoreductase, EC 1.11.1.7) and protoporphyrin-apoperoxidase complex has been shown on the basis of identity of the tryptophan fluorescence parameter at pH 2.0–8.0 and of the circular dichroism spectra of the two proteins. Absorption and fluorescence spectra have been obtained for protoporphyrin in the complex in the pH range 7.0–1.6. A shift in the apparent p K by 4 units has been observed for protonation of the protoporphyrin pyrrolic ring in the complex. From this shift, the dielectric constant has been evaluated for the heme pocket of the peroxidase (approx. 20). Fluorescence quantum yield of protoporphyrin in the complex increased with pH decreasing from 5.0 to 3.5, whereas the spectrum pattern and fluorescence lifetime did not change. The ions, I − and [Fe(CN) 6] −4, peroxidase substrates, did not quench the protoporphyrin fluorescence in the complex at about neutral pH, whereas the quenching markedly enhanced with lowering pH. The bimolecular constant for the I −-quenching of the porphyrin fluorescence in the complex showed a pH-dependence similar to that of the bimolecular rate constant for the reaction of peroxidase compound I with I −. A mechanism for I − oxidation at an acid pH in the presence of peroxidase has been proposed.