A π-Helix Switch Selective for Porphyrin Deprotonation and Product Release in Human Ferrochelatase

Abstract

SUMMARYFerrochelatase (protoheme ferrolyase, E.C. 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 eucaryotic enzyme, details of metal chelation remain unidentified. In this work 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 substratebound 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 π helix that is predicted to have a central role in product release.

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

“…The C ring acetate group apparently plays a major role in inducing the conformational changes that accompany metal ion insertion by moving His207, weakening the grip of the enzyme on the metal and encouraging its insertion into the macrocycle by the exquisitely positioned His145. CbiK His145 is the equivalent to His263 in human ferrochelatase, and in both CbiK and human ferrochelatase this residue moves relatively modestly (27) compared to His207 that is substantially pushed away when SHC binds. The conserved nature of His145 (His263) suggests that this residue plays an important role in metal delivery and together with His207 may also contribute to substrate specificity, product entrapment and enhancement of catalytic rate.…”

Porphyrin Binding and Distortion and Substrate Specificity in the Ferrochelatase Reaction: The Role of Active Site Residues

“…In human ferrochelatase, the porphyrin is rotated by about 100° and buried by an additional 4.5 Å deeper into the active site cleft, relative to the position of N-MeMP bound to the B. subtilis enzyme. 22,23 The structures of the human enzyme also show that the porphyrin binding cleft is in a closed conformation as compared to the porphyrin-free protein, whereas in B. subtilis ferrochelatase the entrance to the cleft is more open after porphyrin binding. Based on these results, it has been suggested that the mode of binding of N-MeMP to B. subtilis ferrochelatase may not reflect the proper substrate-binding mode.…”

“…21 The recently published X-ray structures of the Glu343Lys variant of human ferrochelatase in complex with ppIX, of the lead-inhibited intermediate of the wild-type enzyme, and of the Phe110Ala variant in complex with product (heme) have shown that the porphyrin is rotated by about 100° and buried by an additional 4.5 Å deeper into the active site cleft as compared to the position of N-MeMP in B. subtilis enzyme. 22,23 These differences in porphyrin binding and the apparent differences in the structures of the porphyrin binding pockets of the catalytic antibody and ferrochelatase clearly suggest that the distortions required for the metallation reaction may be imposed by different environments of the substrate.…”

Ferrochelatase forms an oligomeric complex with mitoferrin-1 and Abcb10 for erythroid heme biosynthesis

“…2,3 The Fech-catalyzed reaction involves binding of ferrous iron and protoporphyrin IX substrates to the enzyme, distortion of the protoporphyrin macrocycle followed by porphyrin deprotonation, iron insertion, and product release. 2,4 In mammals, the pathways for iron trafficking and use involve the binding of ferric iron to transferrin for transportation in the circulation and extravascular fluid to cells bearing specific transferrin receptors. Transferrin receptor 1 (TfR1) on erythroid precursors selectively binds diferric transferrin and internalizes it through the receptor-mediated endocytosis of the transferrin-TfR1 complex.…”

“…Ultimately, the Mfrn1-imported iron must be delivered to Fech for heme synthesis. The mechanism of how iron is transferred into Fech in the mitochondrial matrix, however, remains unclear, although it has been suggested that the 2 proteins may form a transient complex 4,9 to facilitate direct iron transfer. Herein we present data demonstrating that Fech forms an oligomeric complex with Mfrn1 and Abcb10 to facilitate mitochondrial ferrous iron transfer for erythroid heme biosynthesis.…”