Human Ferrochelatase: Characterization of Substrate-Iron Binding and Proton-Abstracting Residues † Vera M. Sellers, ‡ Chia-Kuei Wu, § Tamara A. Dailey, § and Harry A. Dailey* ,‡,§ Department of Microbiology, Department of Biochemistry and Molecular Biology, and Center for Metalloenzyme Studies, UniVersity of Georgia, Athens, Georgia 30602-7229 ReceiVed January 3, 2001; ReVised Manuscript ReceiVed May 24, 2001 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. Ferrochelatase (protoheme ferrolyase, EC 4.99.1.1) cata- lyzes the terminal step in heme biosynthesis, the insertion of ferrous iron into protoporphyrin IX to form protoheme IX (heme) (1, 2). In eukaryotes, the enzyme is nuclear- encoded, synthesized in the cytoplasm, and translocated to the mitochondrion where it is proteolytically processed to its mature size of ∼42 kDa (3). Within the mitochondria, ferrochelatase is associated with the inner mitochondrial membrane with the active site facing the mitochondrial matrix (4, 5). Ferrochelatase has been isolated from a variety of sources, and the gene encoding the enzyme has been cloned and sequenced from more than 40 species, including bacteria (6-9), yeast (10), plants (11), and animals (12- 15). Overall, there are fewer than 10% of the residues in the core portion of the enzyme that are identical in all known ferrochelatases. An NO-sensitive [2Fe-2S] cluster has been identified and characterized in recombinant animal ferro- chelatases (15-21), but it is absent in the plant enzymes. Available data on substrate specificity and kinetic param- eters indicate that the mechanism of catalysis is conserved, although substrate specificity varies slightly among species (see ref 1 and references therein). Mammalian ferrochelatase utilizes Fe 2+ as the physiological metal substrate; however, other divalent cations such as Co 2+ and Zn 2+ can serve as alternate substrates. Bacillus subtilis ferrochelatase is unusual in the utilization of Cu 2+ , and not Co 2+ , as an acceptable substrate in vitro (22). In all ferrochelatases, Hg 2+ , Mn 2+ , and Cd 2+ are strong inhibitors while Pb 2+ is a less effective inhibitor (1, 22, 23). Ferrochelatase is thought to utilize an ordered reaction mechanism where iron binds prior to porphyrin (24). Following the binding of the metal ion, distortion of the porphyrin to a nonplanar conformation facilitates porphyrin metalation (25-27). This distortion has been demonstrated by resonance Raman spectroscopy to be a doming (26, 28) or ruffling (29) of the porphyrin macro- cycle. Concomitant with metalation of the macrocycle, two protons are removed from pyrrole nitrogens. Details of the enzyme mechanism along with the nature of the specific amino acids involved in catalysis remain largely unknown. Recently, the three-dimensional structure of B. subtilis ferrochelatase (30) along with the structure of the enzyme-N-methylmesoporphyrin complex were pub- lished (27). These data clearly demonstrated the orientation of the distorted macrocycle in the active site with the center of the porphyrin ring centered directly over the equivalent † This work was supported by grants from the National Institutes of Health (DK32303 and DK35989 to H.A.D.) and by the National Science Foundation Training Group Award to the Center for Metalloenzyme Studies (BIR9413236). * To whom correspondence should be addressed: Department of Biochemistry and Molecular Biology, University of Georgia, Athens, GA 30602-7229. Telephone: (706) 542-2690. Fax: (706) 542-7567. E-mail: hdailey@arches.uga.edu. ‡ Department of Microbiology. § Department of Biochemistry and Molecular Biology and Center for Metalloenzyme Studies. 9821 Biochemistry 2001, 40, 9821-9827 10.1021/bi010012c CCC: $20.00 © 2001 American Chemical Society Published on Web 07/25/2001