The objective of the present proposal is to examine the terminal enzyme in the heme biosynthetic pathway, ferrochelatase (protoheme ferrolyase, E.C. 4.99.1.1.), to determine structural and catalytic information about the enzyme from healthy animal tissue. Bovine liver will be the source of enzyme since a genetic protoporphyria has been described in cattle. Once information is obtained about the enzyme from normal animals, then it will be possible to examine ferrochelatase from animals suffering from protoporphyria so that we may then understand the molecular basis of that disease. The experimental approach outlined herein involves examination of the orientation of ferrochelatase in membranes, definition of possible protein-lipid and protein-protein interactions and study of the catalytic functioning of the enzyme. The data will come largely from chemical modification studies on the purified enzyme as well as examination of interactions in reconstituted liposomal systems and through the use of porphyrin substrate analogs and inhibitors. The ultimate goal of this laboratory is to biochemically characterize the three terminal membrane associated enzymes of this pathway and reconstitute them into liposomes so that they are vectorally organized across the membrane as they are in situ in the mitochondria. The study of the system first in normal animals and then in porphyric animals, along with collaborative studies with clinicians in he field, may lead to the biochemical and molecular understanding of protoporphyria and variegate porphyria.
