Ferrochelatase is the terminal enzyme in the heme biosynthetic pathway and catalyzes the insertion of ferrous iron into protoporphyrin IX in the formation of protoheme IX. Little is known about how substrate iron is acquired and taken up by the enzyme or how the enzyme functions at the cellular level. Here we investigated the putative roles of several amino acid residues in vivo by constructing and analyzing site-directed mutants within the C-terminus, on the protein surface, and inside the active site of human ferrochelatase. In vivo function of the enzyme was assessed by monitoring complementation, cellular growth rates and heme production in a ferrochelatase-deficient strain of Saccharomyces cerevisiae. The data suggest a possible surface site that may be involved in ferrochelatase function. These initial in vivo characterizations may lead to future advances in identifying specific amino acid residues that play a role in ferrochelatase function.
