Ferrochelatase catalyzes the last step in heme biosynthesis: the chelation of a ferrous ion to proto-porphyrin IX, to form protoheme [PMID:2185242, PMID:1704134]. In eukaryotic cells, it binds to the mitochondrial inner membrane with its active site on the matrix side of the membrane.

The X-ray structure of Bacillus subtilis and human ferrochelatase have been solved [PMID:9384565, PMID:11175906]. The human enzyme exists as a homodimer. Each subunit contains one [Fe2S2] cluster. The monomer is folded into two similar domains, each with a four-stranded parallel beta-sheet flanked by an alpha-helix in a beta-alpha-beta motif that is reminiscent of the fold found in the periplasmic binding proteins. The topological similarity between the domains suggests that they have arisen from a gene duplication event. However, significant differences exist between the two domains, including an N-terminal section (residues 80-130) that forms part of the active site pocket, and a C-terminal extension (residues 390-423) that is involved in coordination of the [Fe2S2]cluster and in stabilization of the homodimer. The [Fe2S2] cluster ligands are Cys196, Cys403, Cys406 and Cys411. The experiments with Co(II) binding show that His230 and Asp383 are part of the enzyme active site [PMID:11175906].

Ferrochelatase seems to have a structurally conserved core region that is common to the enzyme from bacteria, plants and mammals. Porphyrin binds in the identified cleft; this cleft also includes the metal-binding site of the enzyme. It is likely that the structure of the cleft region will have different conformations upon substrate binding and release [PMID:9384565].