Xiii. Crystallike Complexes of Apoenzyme with Porphyrins*

Combination of apocytochrome c peroxidase with various porphyrins such as proto-, hemato-, meso-, and deuteroporphyrins IX was investigated. These porphyrins combined with the apoprotein with a molar stoichiometry of 1: 1 to form well defined porphyrin-protein complexes. The complexes were further purified by chromatography on diethylaminoethyl cellulose columns and crystallized by dialysis against distilled water. Spectrophotometric studies of the kinetics of the interaction of the apoenzyme with porphyrins indicated that their reactions were composed of at least two steps, a fast reaction followed by a slow reaction: the former appeared to be a chemical binding of porphyrins to the apoenzyme and the latter a conformational change of the protein moiety caused by the porphyrin binding. The molecular weight and isoelectric point of the protoporphyrin-apoenzyme complex were determined as 4 x lo4 and pH 5.45, respectively. The porphyrin-apocytochrome c peroxidase complexes were found neither to form distinct peroxide compounds in reaction with hydroperoxide nor to show the peroxidase activity. Comparison of these porphyrin-protein complexes with natural and synthetic cytochrome c peroxidases indicated that the iron atom in the prosthetic group is needed for the peroxidase activity but not for the binding of the prosthetic group to the apoenzyme. Light absorption, fluorescence spectra, and heat stability curves of the porphyrin-protein complexes were compared with those of the apoenzyme and natural holoenzyme.