Chelatases: distort to select?

Abstract

Chelatases catalyze the insertion of a specific metal ion into porphyrins, a key step in the synthesis of metalated tetrapyrroles that are essential for many cellular processes. Despite apparent common structural features among chelatases, no general reaction mechanism accounting for metal ion specificity has been established. We propose that chelatase-induced distortion of the porphyrin substrate not only enhances the reaction rate by decreasing the activation energy of the reaction but also modulates which divalent metal ion is incorporated into the porphyrin ring. We evaluate the recently recognized interaction between ferrochelatase and frataxin as a way to regulate iron delivery to ferrochelatase, and thus iron and heme metabolism. We postulate that the ferrochelatase-frataxin interaction controls the type of metal ion that is delivered to ferrochelatase.

Figure I

The relationship between tetrapyrrole biosynthetic pathways. Uroporphyrinogen III is the tetrapyrrole that is common to all tetrapyrrole biosynthetic pathways. 5-Aminolevulinate (ALA), a precursor of uroporphyrinogen III, derives from glycine and succinyl-CoA (in eukaryotes other than plants and the α subgroup of the photosynthetic purple bacteria) or glutamate (in plants and most bacteria). Class I, II and III chelatases are shown in blue, purple and yellow, respectively.

Figure I

Reaction mechanism of porphyrin metalation. (a) Out-of-plane saddling deformation used to describe a nonplanar distortion of the porphyrin macrocycle. Note that the two opposite pyrrole rings with unprotonated nitrogen atoms (blue spheres) point upwards, and the other two pyrrole rings with protonated nitrogen atoms (blue spheres connected to white spheres) point downwards. (b) Steps in the reaction mechanism for incorporation of the metal ion (red) into porphyrin (pyrrole rings, green; unprotonated pyrrole nitrogen atoms, blue; protonated pyrrole nitrogen atoms with protons, yellow) include (i) deformation of the porphyrin ring; (ii) formation of the first metal–porphyrin bond, followed by other ligand-exchange steps leading to formation of a ‘sitting-atop’ complex (in which two pyrrolenine nitrogen atoms coordinate to the metal ion and two protons remain on the pyrrole nitrogen atoms); and (iii) sequential deprotonation of the two pyrrole nitrogen atoms coupled with formation of the metalated porphyrin.

Figure 1

Porphyrin- and metal ion-binding sites in ferrochelatase. (a) Structure of B. subtilis ferrochelatase in complex with the transition-state inhibitor N-methylmesoporphyrin (N-MeMP). The structure is composed of two Rossmann-type domains (green and blue), in which a central four-stranded β sheet is flanked by α helices. A cleft defined by structural elements (red) from both domains accommodates the porphyrin- and metal-binding sites. The inhibitor N-MeMP is shown in the cleft (carbon atoms, yellow; oxygen, red; nitrogen, blue). On binding to the enzyme, porphyrin is distorted and the protein undergoes a conformational transition, during which the cleft is widened. This process is typical of an ‘induced-fit’ mechanism of catalysis. A fully hydrated Mg²⁺ ion is always located ~8 Å from the nitrogen atom of the distorted ring A of the porphyrin (Mg²⁺, green sphere; water, red spheres). (b) Interaction of N-MeMP with amino acids in the substrate-binding cleft of B. subtilis ferrochelatase. The side chains of the residues that contribute directly to the stabilization of porphyrin binding are shown in stick representation. Arg30 and Arg31 are on the surface of the molecule and contribute to neutralization of the negative charge of the propionic acid side chains of the porphyrin. Tyr13, Ile29, His183, Trp230 and Glu264 stabilize the position of the macrocycle in the cleft. The hydroxyl group of Tyr13 is directed towards the center of the ring and coordinates a metal ion in a complex of ferrochelatase with Cu²⁺ inserted into N-MeMP. Tyr26 and Leu43 constitute part of the switch region, which triggers the conformational transition in the enzyme on porphyrin binding. (c) Two metal-binding sites in B. subtilis ferrochelatase. The two sites are shown with a Zn²⁺ ion (gray sphere) and a fully hydrated Mg²⁺ ion (green sphere). The distance between the metal ions is ~7 Å. His183 and Glu264 are among the few residues that are invariant in all protoporphyrin IX ferrochelatases. The side chains of residues Glu272, Asp268 and Glu264 are aligned along a π helix (green) in a line connecting the two metal sites. Only a π helix can provide such an alignment of side chains. The π helix differs from the α helix in that it has 4.1 residues per turn and is stabilized by hydrogen bonds between residues i and i+5 (an α helix has 3.6 residues per turn and hydrogen bonds between residues i and i+4).

Figure 2

Proposed model of modulation of ferrochelatase–frataxin interactions by protoporphyrin. (a) Protoporphyrinogen oxidase (PPO) makes protoporphyrin (PP) available to ferrochelatase (FCH) via a transient interaction (i). Frataxin (Fxn) then delivers Fe²⁺ to ferrochelatase (ii), which promotes its insertion into protoporphyrin to yield heme (iii). (b) In the absence of protoporphyrin, frataxin might donate Fe²⁺ to other proteins (e.g. aconitase) [42] or could incorporate surplus iron in a stable ferrihydrite mineral [43,45].