Abstract Synthetic cytochrome c peroxidases were reconstituted from the apoenzyme and unnatural hemins such as hemato-, meso-, and deuterohemins and 90% 57Fe-enriched protohemin. These synthetic enzymes were crystallized by dialysis against distilled water. On the addition of a stoichiometric amount of hydroperoxide, these synthetic enzymes were converted to stable peroxide intermediates (Complex ES), which retained 2 oxidizing eq derived from hydroperoxide and exhibited an intense electron paramagnetic resonance (EPR) signal of a free radical type at g = 2.004. The synthetic enzymes reacted with various heme ligands such as fluoride, azide, cyanide, and CO to form well defined complexes. The light absorption maxima of these synthetic enzymes and their derivatives were shifted to shorter wave lengths in comparison with the corresponding derivatives of the natural enzyme. However, the over-all patterns of absorption spectra of these derivatives of the synthetic enzymes were very similar to those of the natural enzyme. The EPR spectra of these synthetic enzymes indicated that these enzymes were a mixture of high and low spin compounds at -196°. Cytochrome c peroxidase containing 57Fe-enriched protohemin exhibited slightly broader EPR signals of high and low spin ferric types. However, no hyperfine splitting was observed under the present conditions. The enzymic activities of these synthetic enzymes in the peroxidatic oxidation of ferrocytochrome c, ferrocyanide, and ascorbate were found to be quite similar, indicating that the side chains of porphyrin IX at positions 2 and 4 were not essential for the catalytic activity of cytochrome c peroxidase.