The penultimate step in the heme biosynthetic pathway, the oxidation of protoporphyrinogen IX to protoporphyrin IX, is catalyzed by the enzyme protoporphyrinogen oxidase which is bound to the inner mitochondrial membrane.
The specific aims of this project are to purify this enzyme to homogeneity, characterize both its physical and enzymatic properties, and reconstitute the enzyme in vitro into phospholipid vesicles (liposomes). The possible interactions between protoporphyrinogen oxidase and the terminal enzyme of the pathway, ferrochelatase, will be examined in both an in vitro liposome system where both proteins are bound to a single vesicle and in an in situ type of system composed of isolated mitochondrial inner membrane. The purification will be done using avian erythrocytes and once purified we will characterize the enzymatic properties, determine cofactor requirements, amino acid composition, molecular weight and membrane binding properties. The possible role of specific amino acid residues in interactions with the substrates will be examined by specific chemical modification and after reconstitution into liposomes, lipid-protein interactions will be examined both from a structural and enzymatic viewpoint. Finally the enzyme will be reconstituted into phospholipid vesicles along with purified ferrochelatase and possible protein-protein interactions between these two enzymes will be carefully scrutinized. The ultimate goal of this laboratory is to biochemiclly characterize the three terminal membrane associated enzymes of the pathway and reconstitute them into liposomes so that they are vectorally organized across the membrane as they are in the mitochondria. The study of the system first in normal animals and then in porphyric animals along with collaborative studies with clinicians in the field may eventually lead to the biochemical and molecular understanding of protoporphyria and variegate porphy ria.
