Substrate interactions with human ferrochelatase Amy Medlock, Larkin Swartz, Tamara A. Dailey, Harry A. Dailey, and William N. Lanzilotta* Department of Biochemistry and Molecular Biology, University of Georgia, Athens, GA 30602 Edited by JoAnne Stubbe, Massachusetts Institute of Technology, Cambridge, MA, and approved December 20, 2006 (received for review July 21, 2006) Ferrochelatase, the terminal enzyme in heme biosynthesis, cata- antibody raised against N-methylmesoporphyrin (N-MeMP) IX lyzes the insertion of ferrous iron into protoporphyrin IX to form catalyzes the insertion of metal ions (14–17), the general hypothesis protoheme IX. Human ferrochelatase is a homodimeric, inner has been that N-alkyl porphyrins are transition-state analogs (18– mitochondrial membrane-associated enzyme that possesses an 20). The crystal structure of B. subtilis ferrochelatase with bound essential [2Fe-2S] cluster. In this work, we report the crystal N-MeMP has served as the basis of mechanistic models for ferro- structure of human ferrochelatase with the substrate protopor- chelatases (21). These models, as well as resonance Raman and phyrin IX bound as well as a higher resolution structure of the site-directed mutagenesis studies, assume that the N-MeMP ob- R115L variant without bound substrate. The data presented reveal served in the crystal structure is bound in the active site of that the porphyrin substrate is bound deep within an enclosed ferrochelatase in an orientation that is identical to the spatial pocket. When compared with the location of N-methylmesopor- orientation of the natural substrate/product (22–24). Additionally, phyrin in the Bacillus subtilis ferrochelatase, the porphyrin is it has been assumed that the 36° macrocycle distortion observed in rotated by Ϸ100° and is buried an additional 4.5 Å deeper within the N-MeMP-bound crystal structure represents a catalytic inter- the active site. The propionate groups of the substrate do not mediate that occurs during normal turnover. Data presented herein protrude into solvent and are bound in a manner similar to what provide evidence that these assumptions may not be valid. has been observed in uroporphyrinogen decarboxylase. Further- To provide insight into the interaction of the physiological more, in the substrate-bound form, the jaws of the active site substrate with ferrochelatase, we have determined the structure mouth are closed so that the porphyrin substrate is completely of human ferrochelatase with bound protoporphyrin IX. The engulfed in the pocket. These data provide insights that will aid in human ferrochelatase enzyme used in this investigation was an the determination of the mechanism for ferrochelatase. E343K variant that has a higher affinity for protoporphyrin IX in comparison to the wild-type enzyme. We have also collected heme biosynthesis ͉ protoporphyrin IX ͉ x-ray crystallography ͉ higher resolution data for the previously reported R115L variant metal insertion of human ferrochelatase (25) without bound substrate. The position of the substrate in the E343K variant is distinctly etallated tetrapyrroles are present in most organisms and different from the previously reported orientation of N-MeMP Mparticipate in essential biochemical processes that include in the B. subtilis ferrochelatase (21). In addition to the spatial photosynthesis, oxygen transport, drug metabolism, transcrip- orientation of substrate within the active site of human ferro- tional regulation, NO synthesis, and oxidative phosphorylation. chelatase, this work also shows that the substrate-bound form of Metallation of tetrapyrroles is catalyzed by a group of enzymes the enzyme possesses a ‘‘closed’’ active site conformation that is named chelatases. This group includes, but is not limited to, notably different from the structure of the inhibitor-bound magnesium chelatase, which is essential for chlorophyll produc- B. subtilis ferrochelatase or the structure of the human enzyme tion (1), and ferrochelatase, which is essential for heme produc- without substrate. In both of the latter cases, the active sites are tion (2). Because of the diverse functions of heme, the latter distinctly ‘‘open.’’ These observations provide insight into the enzyme plays a critical role in human health. Human genetic ferrochelatase mechanism of catalysis and inhibition. defects affecting this enzyme have been identified and result in Results the disease erythropoietic protoporphyria (3). Human ferroche- latase, with its [2Fe-2S] cluster (4, 5), represents the convergence General Description of the Overall Structure and Substrate Binding of tetrapyrrole synthesis with iron supply and must play a key role Sites. The crystals of the E343K variant of human ferrochelatase in overall body iron metabolism (6). belong to the triclinic space group P1 (see Table 1), whereas the Ferrochelatases from a variety of sources have been cloned, space group for the initial structure of human ferrochelatase expressed, and characterized to various extents (2, 4), but it is the (containing the amino acid substitution R115L) reported by Wu Bacillus subtilis and mammalian ferrochelatases that have been et al. (25) is orthorhombic (P212121). In the latter case, the studied most extensively. These two enzymes represent the broadest asymmetric unit contained a single biological dimer. The dif- diversity among ferrochelatases examined to date with Ͻ10% ferent crystal symmetry between the substrate-bound form and sequence identity. The B. subtilis protein is a water-soluble, mono- the free enzyme suggests that substantial conformational dif- meric protein with no cofactors (7), whereas the human enzyme is ferences exist between the two forms of the enzyme. Upon an inner mitochondrial membrane-associated homodimer with a phasing and refinement, it was revealed that the asymmetric unit [2Fe-2S] cluster in each subunit (8). Nevertheless, there is clear of the E343K variant of human ferrochelatase contains two structural similarity between these two enzymes. A comparison of the structures reveals a root-mean-square deviation of only 2.4 Å ␣ Author contributions: A.M. and W.N.L. designed research; A.M., L.S., T.A.D., and W.N.L. for the C atoms. The majority of the conserved residues are located performed research; A.M., L.S., and W.N.L. analyzed data; and H.A.D. and W.N.L. wrote the BIOCHEMISTRY in the active site pocket. paper. Since the initial work by DeMatteis’ group that identified and The authors declare no conflict of interest. characterized the ‘‘green pigment’’ in livers of 3,5-dicarbethoxy- This article is a PNAS direct submission. 1,4-dihydrocollidine-treated mice as N-methylprotoporphyrin, Abbreviation: N-MeMP, N-methylmesoporphyrin. there has been considerable interest in N-alkylporphyrins as inhib- Data deposition: The atomic coordinates for the R115L and E343K human ferrochelatase itors of ferrochelatase (9–12). Because of the tight binding com- structures have been deposited in the Protein Data Bank, www.pdb.org (PDB ID codes 2HRC petitive inhibition of ferrochelatase by N-methylprotoporphyrin and 2HRE, respectively). (13), the fact that nonenzymatic metal insertion into porphyrins is *To whom correspondence should be addressed. E-mail: [email protected]. facilitated by macrocycle distortion, and the observation that an © 2007 by The National Academy of Sciences of the USA www.pnas.org͞cgi͞doi͞10.1073͞pnas.0606144104 PNAS ͉ February 6, 2007 ͉ vol. 104 ͉ no. 6 ͉ 1789–1793 Downloaded by guest on September 23, 2021 Table 1. Data collection and refinement statistics R115L E343K Space group P212121 P1 Wavelength 0.98 1.54 Resolution range, Å 40.0–1.7 50.0–2.5 Unique observations 100,275 53,845 Completeness 99.6 (98.2)* 96.7 (93.5) † Rsym,% 0.08 (14.7) 0.08 (28.2) I/␴ 29.1 (5.8) 13.5 (3.4) Unit cell (a, b, c) 88.5, 92.9, 110.4 61.9, 88.3, 93.2 Protein atoms 5,782 11,564 Solvent atoms 601 338 Resolution limits 40.0–1.7 50.0–2.5 Rcryst,% 22.1 21.6 Fig. 2. Structural alignment of the substrate-bound (E343K) and substrate- Rfree,% 24.2 27.8 free (R115L) forms of human ferrochelatase. The substrate-free form of rmsd bonds, Å 0.006 0.008 human ferrochelatase is shown in green, and the substrate-bound form of rmsd angles, ° 1.21 1.61 human ferrochelatase is shown in magenta. Regions of significant movement average B factor, Å2 21.8 31.1 in the substrate-bound form have been highlighted in red for clarity and include residues 90–115, 302–313, and 349–361. The [2Fe-2S] clusters for the *Numbers in parentheses denote values for the outermost resolution shell. substrate-free and substrate-bound forms are shown in yellow and orange, † ϭ͚ ͚ Ϫ͗ ͘ ͚ ͗ ͘ Rsym hkl [ I(Ihkl,I Ihkl )]/ hkl,I Ihkl , where Ihkl is the intensity of an individual respectively. measurement of the reflection with indices hkl and ͗Ihkl͘ is the mean intensity of that reflection. variant possess [2Fe-2S] clusters, as was reported previously for human ferrochelatase. copies of the biological dimer (Fig. 1), for a total of four peptide Comparison of the R115L variant that lacks bound substrate monomers and six protoporphyrin IX molecules. This finding is with the substrate-bound (E343K variant) form reveals that the shown in Fig. 1, which highlights the relative positions of the four enzyme with porphyrin bound possesses a significantly more monomers, six porphyrin molecules, and two detergent mole- ‘‘closed’’ active site conformation. The overall difference be- cules, as well as the [2Fe-2S] clusters. The fact that substrate was tween these two conformations is illustrated in Fig. 2. There is observed in the electron density is of interest because substrate an average deviation of 3.5 Å between the R115L and E343K was not added during the expression, isolation, or crystallization variants for the backbone atoms of residues 90–115. Substantial of the E343K variant. One protoporphyrin molecule is found in differences in the torsion angles of the peptide backbone for the each of the four active sites, and two are located at a noncrys- substrate-bound structure in comparison with the substrate-free tallographic 2-fold axis.