N-Phenylprotoporphyrin IX formation in the hemoglobin-phenylhydrazine reaction. Evidence for a protein-stabilized iron-phenyl intermediate.

Hemoglobin-catalyzed phenylhydrazine oxidation, a model for Heinz-body hemolytic anemias, is known to result in hemoglobin degradation and erythrocyte lysis. The catalytic reaction is shown here to be terminated by inactivation of the prosthetic heme groups after each heme moiety catalyzes the consumption of 6 oxygen molecules and 6 phenylhydrazines, and the formation of 5 benzenes. The phenyl residue not converted to benzene is primarily found, after acidic methanol workup, covalently bound to a nitrogen of protoporphyrin IX. Analogous reactions are observed with substituted phenylhydrazines and, to a lesser degree due to their slower oxidation, with alkylhydrazines. Ortho-substituted phenylhydrazines, however, do not give the N-aryl heme derivatives even though they inactivate the hemoproteins. ESR spin-trapping experiments establish that all of the hydrazines are oxidized to carbon radicals. Direct evidence is provided for the formation of a globin-stabilized heme complex which terminates catalytic activity and which, depending on the conditions of protein denaturation, reverts to heme or is converted to the corresponding N-aryl heme derivative. The globin-stabilized intermediate appears to involve direct coordination of the aryl group through one of its carbon atoms with the prosthetic heme iron moiety.