Arylhydrazines as probes of hemoprotein structure and function

Abstract

The reactions of arylhydrazines (ArNHNH 2) or aryldiazenes (ArNNH) with simple iron porphyrins or with hemoproteins that have relatively open active sites, including hemoglobin, myoglobin, cytochrome P450, chloroperoxidase, catalase, prostaglandin synthase, and indoleamine-2,3-dioxygenase yield σ-bonded aryl-iron complexes. Denaturation of the protein complexes under aerobic, acidic conditions shifts the aryl group to the porphyrin nitrogens and produces mixtures of the four possible N-arylprotoporphyrin IX regioisomers. The regioisomers are obtained in approximately equal amounts if the iron-to-nitrogen shift occurs outside of the protein but the ratio of isomers differs if the rearrangement is controlled by the protein. Only in the case of cytochrome P450 enzymes can the shift be induced to occur without denaturation of the protein. The isomer ratios obtained when the shift occurs in the intact active site provide direct experimental information on the active site topology and dynamics. Topological information has thus been obtained for cytochromes P450 1A1, 1A2, 2B1, 2B2, 2B4, 2B10, 2B11, 2E1, 11A1, 51, 101, 102, and 108. In contrast to hemoproteins with open active sites, conventional peroxidases react with arylhydrazines to give δ- meso-aryl adducts and covalent protein adducts. Reaction with the δ-meso edge but not the heme iron provides key evidence that restricting access of substrates to the ferryl oxygen helps direct the reaction towards peroxidase rather than peroxygenase catalysis.