A pi-helix switch selective for porphyrin deprotonation and product release in human ferrochelatase

Abstract

Ferrochelatase (protoheme ferrolyase, EC 4.99.1.1) is the terminal enzyme in heme biosynthesis and catalyzes the insertion of ferrous iron into protoporphyrin IX to form protoheme IX (heme). Due to the many critical roles of heme, synthesis of heme is required by the vast majority of organisms. Despite significant investigation of both the microbial and eukaryotic enzyme, details of metal chelation remain unidentified. Here we present the first structure of the wild-type human enzyme, a lead-inhibited intermediate of the wild-type enzyme with bound metallated porphyrin macrocycle, the product bound form of the enzyme, and a higher resolution model for the substrate-bound form of the E343K variant. These data paint a picture of an enzyme that undergoes significant changes in secondary structure during the catalytic cycle. The role that these structural alterations play in overall catalysis and potential protein-protein interactions with other proteins, as well as the possible molecular basis for these changes, is discussed. The atomic details and structural rearrangements presented herein significantly advance our understanding of the substrate binding mode of ferrochelatase and reveal new conformational changes in a structurally conserved pi-helix that is predicted to have a central role in product release.

Figure 1

Occupancy of the active site for WT1 and R115L. (a) Overlay of cholate residues and the side chains of H263, R115 and M76 of the WT1 model with the model previously reported for the R115L variant of human ferrochelatase. (b) Heme molecule modeled into the density present in the active site of monomer B of the WT1 data with side chains of M76 below and H263 above the macrocycle ring. All atoms in the WT1 model are represented as sticks with nitrogen, oxygen, carbon, sulfur, and iron atoms colored blue, red, tan, cyan, and black, respectively. All atoms in the model previously reported for R115L variant are colored green in (a). In all cases the 2F_o-F_c composite omit map was generated using the simulated annealing protocol and is contoured at 1σ (purple and green cage in panels a and b, respectively).

Figure 2

Substrate binding to the E343K variant of human ferrochelatase. (a) Schematic representation of protoporphyrin IX. (b) Cartoon representation of the overall fold of the protoporphyrin-bound structure of the E343K variant showing the position of the substrate relative to the π helix (colored dark blue) in human ferrochelatase. (c) Wall-eyed stereo view of the composite omit map for the substrate and residues near the substrate in the π helix. The atoms are represented as sticks and are colored blue, red and tan for nitrogen, oxygen, and carbon, respectively. The 2F_o-F_c composite omit map is contoured at 1σ (green cage) and was generated using the simulated annealing protocol with 7 % of the model being omitted per cycle.

Figure 3

Model of protoporphyrin IX in the active site and composite omit map of monomer A for the E343K data highlighting residues within van der Waals contact of the substrate. (a) View of the porphyrin macrocycle and nearby residues on the H263 side of the pocket. (b) View of the porphyrin in the active site looking at the substrate from the same side of the substrate as residue M76. The atoms are represented as sticks and are colored blue, red, yellow and tan for nitrogen, oxygen, sulfur and carbon, respectively. For clarity the 2F_o-F_c omit map is contoured at 1σ around the substrate only and was generated as described in the legend to Figure 2.

Figure 4

Wall-eyed stereo diagram for the model and 2F_o-F_c composite omit map (green cage) showing porphyrin macrocycle and the unwound π helix observed in (a) the lead-inhibited ferrochelatase and (b) the protoheme-bound F110A variant. Also visible in are the axial ligands for the porphyrin-bound metal: acetates in (a) and imidazole (labeled) and bicarbonate in (b). The models are shown in ball and stick format with the nitrogen, oxygen, carbon, lead, and iron atoms colored blue, red, tan, yellow, and black, respectively. The 2F_o-F_c composite omit map is contoured at 1σ and was generated as described in the legend to Figure 2.

Figure 5

Electrostatic surface potential showing the active site region for the (a) wild type, (b) the substrate bound, and (c) heme bound human ferrochelatase. For clarity, the upper lip and π helix regions are highlighted. The figure was generated with PYMOL.

Figure 6

Cartoon representation of the structural alignment of the free, substrate-bound and product-bound forms of human ferrochelatase. (a) Cartoon overlay of the wild-type, substrate-free structure in cream color. Areas of significant movement are highlighted in color. The upper lip of the active site that is altered in spatial orientation in the E343K substrate-bound variant relative to the wild-type enzyme structure is shown in green. The unwound π helix segment of the F110A product-bound variant enzyme is shown in red. Bound protoporphyrin IX is shown in brick and highlighted using dots to denote the electron density. The [2Fe-2S] cluster is shown as yellow and blue spheres. (b) Enlarged view of the bound protoporphyrin IX (brick) and bound heme (purple) in the same spatial orientation as shown in the cartoon model. The movement of the protoporphyrin/protoheme IX propionate 6, which is on the corner of the macrocycle in this orientation, is highlighted by the grey arrow.

Figure 7

Model for the possible role of structural changes in putative protein-protein interactions. Cartoon representation of how human ferrochelatase (Fc) might interact with protoporphyrinogen oxidase (PPO) during binding of protoporphyrin IX. Following iron insertion the π helix is unwound and a new binding surface is presented for the release of protoheme to a currently unidentified heme carrier protein (HCP). The inner mitochondrial membrane is shown as solid lines.