The apo-enzymes of porphobilinogen oxygenase and horseradish peroxidase were reconstituted with hemin IX, deuterohemin IX, 2,4-diacetyldeuterohemin IX, 2-vinyl-4-deuterohemin IX and hemin I. The apoproteins did not reconstitute with the dimethyl or diethyl esters of hemin IX. The native enzymes and the synthetic hemoproteins showed similar oxygenase activities toward porphobilinogen in the presence of dithionite and oxygen. They also showed peroxidase activity in the presence of H2O2, which was affected by the side-chain substitution pattern of the hemes. Oxygenase activities, however, were not affected by the heme structure. Iron chelators completely inhibited the oxygenase, but not the peroxidase activities. The EPR spectra of the native and synthetic porphobilinogen oxygenase showed that dithionite reduction produced a rapid disappearance of the high-spin heme-iron signal at g= 6.0. It reappeared 1 min later but the enzyme retained its catalytic activity. The changes in the EPR spectra could be correlated with the biphasic kinetics of the oxygenase reaction which was very fast during the first minute and then decreased to a half-value rate. The oxygenase reaction was inhibited by addition of superoxide dismutase during the fast rate phase, but not during the slower phase. These results could be explained by the formation of a superoxide anion during the first minute of the oxygenase reaction, after which a protein-stabilized radical (g= 2.0) is generated (very likely a tyrosyl radical). The latter then oxidizes the substrate porphobilinogen and facilitates its reaction with O2 to give oxopyrrolenines.