Human Ferrochelatase:  Characterization of Substrate−Iron Binding and Proton-Abstracting Residues

Abstract

The terminal step in heme biosynthesis, the insertion of ferrous iron into protoporphyrin IX to form protoheme, is catalyzed by the enzyme ferrochelatase (EC 4.99.1.1). A number of highly conserved residues identified from the crystal structure of human ferrochelatase as being in the active site were examined by site-directed mutagenesis. The mutants Y123F, Y165F, Y191H, and R164L each had an increased K(m) for iron without an altered K(m) for porphyrin. The double mutant R164L/Y165F had a 6-fold increased K(m) for iron and a 10-fold decreased V(max). The double mutant Y123F/Y191F had low activity with an elevated K(m) for iron, and Y123F/Y165F had no measurable activity. The mutants H263A/C/N, D340N, E343Q, E343H, and E343K had no measurable enzyme activity, while E343D, E347Q, and H341C had decreased V(max)s without significant alteration of the K(m)s for either substrate. D340E had near-normal kinetic parameters, while D383A and H231A had increased K(m)s for iron. On the basis of these data and the crystal structure of human ferrochelatase, it is proposed that residues E343, H341, and D340 form a conduit from H263 in the active site to the protein exterior and function in proton extraction from the porphyrin macrocycle. The role of H263 as the porphyrin proton-accepting residue is central to catalysis since metalation only occurs in conjunction with proton abstraction. It is suggested that iron is transported from the exterior of the enzyme at D383/H231 via residues W227 and Y191 to the site of metalation at residues R164 and Y165 which are on the opposite side of the active site pocket from H263. This model should be general for mitochondrial membrane-associated eucaryotic ferrochelatases but may differ for bacterial ferrochelatases since the spatial orientation of the enzyme within prokaryotic cells may differ.

Evolution in a family of chelatases facilitated by the introduction of active site asymmetry and protein oligomerization

“…Moreover, site directed mutagenesis of the histidine residue is also consistent with this hypothesis (22). Nonetheless, evidence of metal binding to an area behind the active site in human protoporphyrin ferrochelatase has led to a controversial idea that the metal ion may travel to the active site via a channel spanning a distance of around 20 Å (20,23). Thus, there is still some debate on not only how the metal ion binds to the chelatase but also how the metal ion is delivered to the enzyme.…”

Probing the Active Site Loop Motif of Murine Ferrochelatase by Random Mutagenesis

“…In the wild-type model, the N-terminal loop residues Gln 248 and Ser 249 were buried deeply inside the active site cavity and in very close proximity (about 4 Å) to His 209 and Glu 289 , which were proposed to be essential for catalysis (31)(32)(33)(34). Lys 250 was solvent-exposed, with the side chain projecting to the protein exterior and in close contact with the Gln 260 residue.…”

Amyloid-β peptide binds with heme to form a peroxidase: Relationship to the cytopathologies of Alzheimer’s disease

“…␦-Aminolevulinic acid synthase (ALAS) catalyzes the condensation of succinyl-CoA with glycine, producing ALA (13), which is the first and rate-limiting step in heme synthesis. Ferrochelatase, located in the inner membrane of the mitochondria, inserts iron into protoporphyrin IX to produce heme (14,15), which then reaches the cytosol to form the regulatory heme. Regulatory heme binds to the hemeregulatory motif in specific proteins.…”