STRUCTURE-ACTIVITY-RELATIONSHIPS IN PORPHOBILINOGEN OXYGENASE AND HORSERADISH-PEROXIDASE - AN ANALYSIS USING SYNTHETIC HEMINS

The apo-enzymes of porphobilinogen oxygenase and horseradish peroxidas e were reconstituted with hemin IX, deuterohemin IX, 2,4-diacetyldeute rohemin 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 oxygenas e 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 structu re. Iron chelators completely inhibited the oxygenase, but not the per oxidase activities. The EPR spectra of the native and synthetic porpho bilinogen oxygenase showed that dithionite reduction produced a rapid disappearance of the high-spin heme-iron signal at g = 6.0. It reappea red 1 min later but the enzyme retained its catalytic activity. The ch anges in the EPR spectra could be correlated with the biphasic kinetic s of the oxygenase reaction which was very fast during the first minut e and then decreased to a half-value rate. The oxygenase reaction was inhibited by addition of superoxide dismutase during the fast rate pha se, 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 oxidize s the substrate porphobilinogen and facilitates its reaction with O2 t o give oxopyrrolenines.