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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.
Insertion of a specific divalent metal ion into a tetrapyrrole provides the basis for a multitude of cellular functions. The resulting metalated tetrapyrroles constitute the centerpiece for proteins involved in cellular processes ranging from oxygen and electron transport, signal transduction, photosynthesis, and sulfur and nitrogen metabolism to molecular rearrangements, methyl-group transfer reactions and regulation of transcription and translation [1–4]. Metalated tetrapyrroles such as the cofactors and prosthetic groups heme, chlorophyll, cobalamin (vitamin B12) and coenzyme F430 have a centrally chelated metal ion and arise from a common tetrapyrrole, uroporphyrinogen III. The chemical nature of the chelated metal ion varies; for example, Fe2+ is present in heme, Mg2+ in chlorophyll, Co2+ in cobalamin and Ni2+ in coenzyme F430 [5–9].
Essential to the catalytic insertion of metal ions into various tetrapyrroles is a divergent group of enzymes called chelatases [5–9], which ensure specificity of the metal ion in response to cellular requirements (Box 1). Although a model of the general reaction mechanism of porphyrin metalation has emerged [10–15], how chelatases bestow metal ion selectivity and specificity on porphyrin metalation remains unresolved.
Tetrapyrroles are organic molecules that contain four five-membered heterocyclic (pyrrole) rings, linked in a cyclic or linear array. Heme, chlorophyll, cobalamin (vitamin B12), siroheme and coenzyme F430 belong to a family of prosthetic groups that are characterized by their tetrapyrrole-derived nature and contain a central, complexed metal ion: Fe2+ in heme and siroheme, Mg2+ in chlorophyll and bacterio-chlorophyll, Co2+ in cobalamin, and Ni2+ in coenzyme F430 [5,6,8,9]. All of these molecules are derived from a common tetrapyrrole, uroporphyrinogen III (Figure I), and require the function of specific enzymes or chelatases for the insertion of their metal ion (Figure I).
On the basis of structural, functional and genetic studies and results from genome sequencing projects [6,24], three main classes of chelatases are recognized: (i) ATP-dependent heterotrimeric chelatases, including protoporphyrin IX magnesiumchelatase (ChlH-I-D or BchlH-I-D) involved in chlorophyll or bacteriochlorophyll biosynthesis , and aerobic cobaltochelatase (CobN-S-T) associated with cobalamin biosynthesis ; (ii) ATP-independent chelatases, including protoporphyrin IX ferrochelatase (HemH) involved in heme biosynthesis [5,16,33], sirohydrochlorin ferrochelatase (SirB) associated with siroheme biosynthesis [6,7], and anaerobic sirohydrochlorin cobalto-chelatases (CbiK and CbiX) involved in cobalamin biosynthesis ; and (iii) multifunctional homodimeric chelatases associated with siroheme biosynthesis, including siroheme synthase (CysG) , and precorrin-2 dehydrogenase and sirohydrochloride ferrochelatase (Met8p) in yeast . The enzymes in the third class house two enzymatic activities: dehydrogenase and chelatase [8,24].
Although there is no sequence similarity among chelatases and no significant sequence similarity (<12% overall sequence identity) between members of the ATP-independent chelatase class, the latter have similar overall topology and adopt a similar fold [5,6,16]. The ATP-independent chelatases possess two α/β domains with a similar Rossmann-type fold; the active site cleft is located between the N- and C-terminal domains and structural elements are contributed from both domains [5,6,33]. The different chelatases are thought to have similar enzymatic mechanisms, involving distortion of the tetrapyrrole, abstraction of two protons from the tetrapyrrole-derived substrate, and exposure of the pyrrole nitrogen atoms to the incoming metal ion substrate [6,16].
Among the chelatases, protoporhyrin IX ferrochelatase (protoheme ferrolyase, EC 126.96.36.199; hereafter called ferrochelatase) represents the most-investigated enzyme (Figure 1a). It catalyzes the insertion of Fe2+ into protoporphyrin IX , the terminal step of the heme biosynthetic pathway. Distortion of porphyrin, which follows porphyrin binding to ferrochelatase, is a salient feature in the catalytic mechanism of this enzyme and embodies an ‘induced-fit’  mechanism of catalysis.
Here we focus on the catalytic reaction of ferrochelatase as a model for all chelatases by exploring the interplay between distortion of the porphyrin substrate and metal ion insertion. We propose that chelatases, by differentially distorting the porphyrin substrate, modulate which metal ion is incorporated. In addition, we assess emerging roles associated with ferrochelatase – namely, its potential interaction with frataxin – and evaluate the significance of this protein–protein interaction with regard to the regulation of iron homeostasis and heme biosynthesis. We propose that protoporphyrin modulates the interaction between ferrochelatase and iron-loaded frataxin.
Distortion of porphyrin has long been recognized as a crucial step in the incorporation of metal ions into porphyrin molecules (Box 2). The crystal structure of ferrochelatase complexed with N-methylmesoporphyrin (N-MeMP), a potent inhibitor of ferrochelatase (dissociation constant, Kd≈10 nM) that mimics a strained substrate [18–20], shows that porphyrin binds in a deep cleft between two surfaces: one hydrophobic and one hydrophilic (Figure 1b). When used as a hapten, non-planar N-MeMP induces the formation of antibodies that catalyze the insertion of divalent metal ions into mesoporphyrin [19,21,22].
More than 30 years ago, Hambright and Chock  proposed a mechanism for metal chelation involving formation of an outer-sphere complex between free-base porphyrin and a metal ion, followed by deformation of the porphyrin nucleus to generate an appropriate configuration for complexation of the metal ion (Figure I). Accordingly, the conformer would have two of the opposite pyrrole rings and lone-pair orbitals of the respective pyrrole nitrogen atoms pointing upwards, and the other two pyrrole rings would be pointing downwards with regard to the mean porphyrin plane (Figure Ia). Hambright and Chock  suggested that the coordinating ability of the lone pairs would be enhanced in this conformation, because the lone pairs would point away from the center of the macrocycle and the apparent acidity of the protons would be increased owing to elimination of the N–H tautomerism . In summary, porphyrin distortion would heighten metal chelation, and deformation – rather than dissociation – would control porphyrin metalation .
Subsequent studies in solution and with enzymatic systems led to the formulation of a general reaction mechanism for the metalation of porphyrins [12–15]. The steps for the mechanism include, first, deformation of the porphyrin ring; second, outer-sphere association of the solvated metal ion and the porphyrin; third, exchange of a coordinated solvent molecule with the first pyrrolenine nitrogen atom; fourth, chelate-ring closure with liberation of additional solvent molecules to form the ‘sitting-atop complex’; fifth, first deprotonation of a pyrrole nitrogen atom; and last, deprotonation of the second pyrrole nitrogen with formation of the metalated porphyrin (Figure Ib).
Thus, induction of porphyrin distortion should be a reasonable role for a catalyst of metal ion insertion. Indeed, Lavallee  reasoned that ferrochelatase should catalyze porphyrin metalation mainly by facilitating porphyrin distortion and keeping the outer-sphere complex intact for the complete reaction to occur. Recent calculations of the energetics associated with porphyrin distortion have shown that this step forms an integral part of the course of the ferrochelatase-catalyzed reaction . Sigfridsson and Ryde  used combined quantum mechanical and molecular mechanical calculations to examine the energetics of porphyrin deformations in free porphyrins, ferrochelatase and the porphyrin–ferrochelatase complex. Their results clearly demonstrate that ferrochelatase-induced distortion of free-base protoporphyrin IX – the physiological substrate of ferrochelatase – is thermodynamically favorable. Their model supports a saddled structure for the porphyrin, in which two opposite pyrrole rings are slightly tilted upwards and the other two pyrrole rings are slightly tilted downwards.
Antibody 7G12, the result of five somatic mutations accumulated during affinity maturation of the germline precursor antibody, is an example of such a catalytic antibody [19,21,22]. Similar to ferrochelatase, antibody 7G12 has a porphyrin-binding cleft at the junction of two surfaces with different hydrophobicities [19,22]. Whereas binding of N-MeMP to Bacillus subtilis ferrochelatase leads to a conformational transition in ferrochelatase with widening of the binding cleft in a characteristic ‘induced-fit’ reaction mechanism , however, antibody 7G12 binds porphyrin in a ‘lock-and-key’ fashion without undergoing a conformational transition [19,21]. These findings indicate that, in contrast to ferrochelatase, the binding site of antibody 7G12 is rigid and pre-organized to bind a distorted porphyrin.
Although ferrochelatase and antibody 7G12 are both effective catalysts of porphyrin metallation, the specific interactions that lead to distortion of the macrocycle structure are almost certainly very different. Thus, in ferrochelatase, porphyrin rings B, C and D are held in a vice-like grip through interactions with conserved amino acid side chains (Figure 1b). For ring A, which is tilted, it is difficult to identify a single residue that might be responsible for this distortion. The binding pocket for this pyrrole is created by several residues, which, together with the strain generated by the tight binding of rings B, C and D, presumably produce the force necessary for tilting. Because tetrapyrroles are complex conjugated systems, however, tilting one of the rings will inevitably distort the other rings.
In antibody 7G12, N-MeMP and mesoporphyrin are bound in a cleft formed by the complementarity-determining regions (CDRs) L2 and L3 on one side and CDR H3 on the other [19,22]. The side chain of Asp100L (Asp100 of the light-chain variable region of the 7G12 antibody) has an important role in interactions with the porphyrin. In the complex with mesoporphyrin (Protein Data Bank accession code 1NGW), the carboxylic oxygen of Asp100L points towards the center of the ring and is located within hydrogen-bonding distance of the nitrogen atoms of rings A (2.8 Å), B (2.5 Å) and C (2.9 Å) [19,22]. The distance to the nitrogen atom of ring D is 3.7–3.8 Å, which is too long for a hydrogen bond.
A similar asymmetric interaction of a polar side chain with the pyrrole nitrogen atoms is adopted in macrocycle distortion by other proteins. For example, in the complexes of uroporphyrinogen decarboxylase, an enzyme of the heme biosynthetic pathway, with the coproporphyrinogen I and III isomers , the carboxylic oxygen of Asp86 shows an asymmetric distribution of distances to the nitrogen atoms of the four pyrrole rings: 2.8 Å, 2.9 Å, 3.1 Å and 3.4 Å. The tetrapyrrole lies against a surface built by conserved hydrophobic residues and adopts a domed conformation in which one pyrrole ring (with the longest distance to Asp86) is tilted out of the plane formed by the nitrogen atoms of the other three pyrroles. Phillips et al.  conclude that coordination of Asp86 to the pyrrole nitrogen atoms is one of the main factors that contributes to distortion of the tetrapyrrole ring.
We suggest that Asp100L of the catalytic antibody has a similar function in distortion of the bound mesoporphyrin. A comparable mode of porphyrin distortion, involving a conserved aspartate residue, might be used by Met8p, a bifunctional dehydrogenase and ferrochelatase. The Met8p aspartate is located in the potential substrate-binding cleft and seems to perform dual functions in dehydrogenation and metalation . Although anaerobic cobalt chelatase is structurally homologous to ferrochelatase (Box 1), the absence of structural information on substrate binding prevents a reliable analysis of the mechanism of substrate distortion in this enzyme. Nevertheless, the above analysis of porphyrin binding indicates that different binding modes probably result in different types of ring distortion, which in turn affect the reactivity of the macrocycle and its chelating ability.
Porphyrin binding and distortion in Saccharomyces cerevisiae and murine ferrochelatases and in antibody 7G12 have been directly assessed using resonance Raman spectroscopy [21,25–27]. In both enzymes and the antibody, porphyrin binding activates several assigned out-of-plane vibrational modes of the macrocycle. The frequency shifts in the structure-sensitive lines of the bound porphyrins relative to the unbound porphyrins reflect the type of distortion that is undergone by the porphyrin macrocycle. Significantly, γ15, a saddling-symmetry (B2u) out-of-plane mode, emerges with the binding of free-base protoporphyrin to murine ferrochelatase  and to the catalytic antibody . A saddling deformation has also been identified as a chief component of the nonplanar distortion of either N-MeMP or Cu-N-MeMP bound to B. subtilis ferrochelatase in the crystal structures of these complexes .
In fact, the normal-coordinate structural decomposition of the porphyrins contained in the crystal structures accounts for how the different symmetric out-of-plane deformations (ruffled, saddled, domed, x- and y-waved, and propellered structures) contribute to the porphyrin distortion . The main calculated contributions to the distortion of N-MeMP and Cu-N-MeMP bound to B. subtilis ferrochelatase are saddling, followed by x-waving, ruffling and doming . We reiterate that saddling is an out-of-plane deformation that exposes both the protons and the lone pairs of the nitrogen atoms of the porphyrin macrocycle, in keeping with a porphyrin arrangement for metal insertion.
Although the physiologically relevant reaction catalyzed by ferrochelatase is insertion of Fe2+ into the porphyrin ring, the enzyme can chelate other divalent metal ions (e.g. Zn2+, Co2+ and Cu2+) . Curiously, in contrast to most ferrochelatases, the B. subtilis enzyme can chelate Cu2+ but not Co2+. Thus, understanding the metal-binding site was cited early on as a significant goal in defining the metal substrate specificity and the reaction catalytic mechanism .
Different spectroscopic techniques (e.g. extended X-ray absorption fine structure and Mössbauer), X-ray crystallography and site-directed mutagenesis have been used to characterize the metal-binding sites of ferrochelatase [5,29–35]. Mössbauer and X-ray absorption spectroscopies have indicated that ferrochelatase has an ionic coordination environment for Fe2+ with six nitrogen- and/or oxygen-containing ligands [29,30]. These results are consistent with a proposal, derived from kinetic studies of site-directed variants of yeast and human ferrochelatases, that a conserved histidine residue (His235 in yeast and His263 in human) is involved in binding the metal substrate [31,32]. The B. subtilis and yeast ferrochelatase crystal structures both support the coordination of a metal ion to the corresponding histidine (His183 in B. subtilis ferrochelatase)  (Figure 1c).
In particular, the crystal structures of yeast ferrochelatase complexed with either Co2+ or Cd2+ have revealed that three conserved amino acids (His235, Glu314 and Ser235) are involved in coordination of the metal ion . His235 is the ligand at the shortest distance from the metal ion, thereby supporting its proposed role as the main metal-binding residue . This view, however, is not undisputed. Analysis of the crystal structure of Co2+-reacted human ferrochelatase revealed a metal-binding site that is located ~20 Å from His263 on the opposite side of the porphyrin-binding cleft . Wu et al.  proposed a model in which the Fe2+ ion, after initially binding at this site (His230 and Asp383 in human ferrochelatase), translocates to the porphyrin-binding site through a conduit of conserved residues. In this model, the conserved active site histidine (His263 in human ferrochelatase) is responsible for abstraction of the porphyrin proton [16,35].
Determination of the structures of B. subtilis ferrochelatase complexed with either Cd2+ or Zn2+ has led to the clear identification of two metal-ion-binding and possibly interacting sites: one located at the surface of the molecule; and one located in the porphyrin-binding cleft, close to the distorted porphyrin ring A . In fact, the nitrogen of ring A is directed towards His183 and Glu264  (Figure 1c). The two metal-binding sites are located 7 Å apart, and the affinity of a metal ion towards each of them depends on the presence of a metal ion at the other site .
Lecerof et al.  proposed that a metal ion, by coordinating with acidic residues arranged along the helical edge of a π helix, would be shuttled from the site at the surface to the inner site. Similar to several other proteins in which π-helix residues function to coordinate metal ions required for enzymatic activity, the residues along the π helix in ferrochelatase might form a channel that connects the interior of the active site to the protein exterior . Lecerof et al.  also advanced the hypothesis that channeling of the metal ion would require an exchange of ligands, initially between the solvent to which the metal is coordinated and the π-helix acidic residues, and ultimately between the protein ligands and the pyrrole nitrogen atoms, thereby culminating with insertion of the metal ion into the porphyrin macrocycle.
In non-enzymatic metalation, the relative order of divalent metal ion insertion into the porphyrin ring, Cu2+>Zn2+>Mn2+~Co2+>Fe2+>Ni2+>Cd2+Mg2+, matches the rates of solvent exchange for the ions . Recently, Shen and Ryde  assessed the importance of ligand exchange in the non-enzymatic incorporation of either Fe2+ or Mg2+ into a porphyrin ring and found that the ligand-exchange step is rate limiting for the reaction. The estimated activation energies associated with this step were ~51 and 72 kJ/mol for Fe2+ and Mg2+, respectively. Metal desolvation and the role of ligand exchange in the catalytic mechanism of ferrochelatase or any other chelatase has been addressed only recently by Shipovskov et al. , who suggested that ferrochelatase accelerates the rate of ligand exchange for the metal.
For non-enzymatic porphyrin metalation, Shen and Ryde  also proposed that deprotonation might be facilitated by hydrogen-bond networks, which translocate the proton away from the ‘sitting-atop complex’ (Box 2). Although the nature of the catalytic base involved in proton abstraction in ferrochelatase or any other chelatase remains to be determined, it is expected that any amino acid involved in deprotonatation will be located on the side of the porphyrin ring opposite to the metal ion. It is also possible that the proton is quickly released to the solvent after being removed by an amino acid, enabling the same amino acid to bind the metal ion before its insertion into the porphyrin. Alternatively, the porphyrin ring might be directly deprotonated by water molecules present in the porphyrin-binding cleft .
Independent experimental approaches have provided unequivocal evidence that porphyrin binding to ferrochelatase induces distortion of porphyrin [20,27]. Although axial ligation is not crucial for porphyrin distortion in the active site of ferrochelatase [20,27], however, the catalytic antibody  and probably Met8p  use axial ligation to induce distortion in the tetrapyrrole macrocycle. Given that the porphyrin-binding cleft is a common structural feature among chelatases [5,33,35], can differences in the mode of porphyrin binding and distortion explain the variation observed in metal specificity and catalytic activity? A strong argument in support of such a possibility is the high conservation of the porphyrin-binding pocket in ferrochelatases, which can be easily seen when the structures of B. subtilis, Homo sapiens and S. cerevisiae ferrochelatases are compared. The conserved residues on one side of the porphyrin ring (Ala182, His183, Ser184, Leu185, Pro186 and Met187 in B. subtilis ferrochelatase) can be superimposed on the corresponding residues of the yeast structure (Ala234, His235, Ser236, Leu237, Pro238 and Met239) with a root mean square deviation of 0.53 Å for the Cα atoms. This high degree of conservation in the cleft suggests that the mode and degree of porphyrin distortion are important parameters of the enzymatic reaction.
Of relevance, the degree of a specific nonplanar deformation of porphyrin contributes to the catalytic efficiency of ferrochelatase. Strikingly, the activation of an out-of-plane porphyrin vibration, γ15, correlates with the catalytic efficiency of antibody 7G12 . We propose that subtle structural differences among the active sites impose distinct degrees of porphyrin distortion and selective incorporation of the metal ion. Compelling evidence in support of this hypothesis indicates that metalation of N-MeMP by Cu2+ is more efficient than that by Zn2+. This contrasts with the lower Michaelis constant for metalation of protoporphyrin IX by Zn2+(17 μM) as compared with Cu2+(170 μM) . Thus, it seems that the different types of distortion of N-MeMP and protoporphyrin IX, when bound to ferrochelatase, affect the specificity of the enzyme towards the metal ion.
The chelatase-induced distortion of the porphyrin substrate substantiates particularly well the hypothesis formulated by Jencks  that ‘the induction of strain in the substrate, the enzyme, or both plays a significant role in bringing about the observed specific rate acceleration’. The induction of strain in the enzyme–substrate complex decreases the activation energy of the reaction, but the cost of strain energy is a decrease in substrate-binding energy (i.e. the ‘Circe effect’) [38,39]. Thus, the relationship between ferrochelatase catalytic efficiency and the extent of the saddling undergone by porphyrin is consistent with the idea that catalysis involves straining of the substrate and evolution of the binding energy to lower the activation energy of the reaction. Furthermore, such a change in porphyrin and protein conformations on complex formation  exemplifies an ‘induced-fit’ reaction mechanism.
The protein conformational transition on porphyrin binding to ferrochelatase might also have an additional function. Protoporphyrinogen IX oxidase, the enzyme preceding ferrochelatase in the heme biosynthetic pathway, has been proposed to form a complex with ferrochelatase across the inner mitochondrial membrane [40,41]. This possibly transient complex should facilitate the direct channeling of protoporphyrin IX to ferrochelatase. Thus, the change in protein conformation that follows porphyrin binding might control the affinity of ferrochelatase towards protoporphyrinogen IX oxidase, resulting in dissociation of the complex.
Under physiological conditions, efficient delivery of Fe2+ to ferrochelatase requires mechanisms to overcome both the limited bioavailability and the potential toxicity of iron. The mitochondrial protein frataxin can either make Fe2+ available to other proteins [42–44], thereby acting as a general iron chaperone, or convert surplus iron to a redox-inactive mineral , thereby providing a mechanism for iron detoxification. Growing evidence supports the hypothesis that frataxin is a facilitator of iron metabolism , promoting not only the biosynthesis and repair of iron–sulfur clusters [42,47] but also the biosynthesis of heme [48,49]. In S. cerevisiae knockout mutants lacking frataxin, ferrochelatase catalyzes the formation of Zn2+-protoporphyrin but not heme. Thus, although ferrochelatase is catalytically competent, it cannot catalyze the insertion of Fe2+ into protoporphyrin, suggesting that frataxin has an important physiological role in controlling the availability of mitochondrial Fe2+ to ferrochelatase .
Frataxin is inferred to be an iron donor in heme biosynthesis with an estimated Kd of 1.7×10−8 to 4×10−8 M for ferrochelatase [50,51]. The ferrochelatase–frataxin interaction has been confirmed by in vitro ferrochelatase activity assays, which have established that Fe2+-loaded frataxin supports heme formation by providing ferrochelatase with Fe2+ in the presence of atmospheric oxygen without reducing agents [43,45]. Determination of the crystal structure of human frataxin identified a conserved and predominantly hydrophobic region on the surface of the protein that was postulated to mediate protein–protein interactions . More recently, He et al. , using NMR spectroscopy to determine the yeast frataxin solution structure, identified the frataxin helical plane as the possible ferrochelatase-binding surface.
Similar to the role of citrate in stabilizing the interaction between frataxin and aconitase , protoporphyrin might stabilize the frataxin–ferrochelatase complex and, in so doing, facilitate the ferrochelatase-catalyzed reaction and formation of heme. The relative concentrations of protoporphyrin and other substrates (citrate, sulfur) might regulate the extent to which frataxin delivers iron for heme and/or iron–sulfur cluster synthesis. Protoporphyrin probably becomes available to ferrochelatase through a complex formed, perhaps even transiently, with protoporphyrinogen oxidase (Figure 2). Protoporphyrin would then induce the interaction between ferrochelatase and iron-loaded frataxin, which in turn would deliver Fe2+ to ferrochelatase for insertion into the protoporphyrin ring to give heme.
In summary, the presence of frataxin would promote the use of Fe2+ by ferrochelatase. In a situation where frataxin is absent or defective, such as in the yeast knockout model or in individuals affected with the neurodegenerative disease Friedreich’s ataxia, ferrochelatase would not have Fe2+available and would use other divalent metal ions such as Zn2+ as a metal ion substrate [48,49]. We therefore propose that protoporphyrin, along with modulating the interaction between ferrochelatase and protoporphyrinogen oxidase, modulates the frataxin–ferrochelatase interaction.
We postulate that the distortion of porphyrin adopted during porphyrin binding to a chelatase, in addition to enhancing the reaction rate, controls the selection of the metal ion inserted into the porphyrin macrocycle. As a determinant of metal ion substrate specificity in chelatase-catalyzed reactions, the extent and type of porphyrin distortion adds a new dimension to the functional value of porphyrin deformation.
The interaction between ferrochelatase and frataxin assumes a crucial role in the ferrochelatase reaction in the cell, because frataxin, functioning as an iron chaperone, provides ferrochelatase with Fe2+. We propose that frataxin interacts with ferrochelatase in a porphyrin-dependent manner, delivering Fe2+ according to cellular requirements. Finally, we postulate that protoporphyrin modulates the interaction between ferrochelatase and iron-loaded frataxin.
Future research on the type and degree of porphyrin distortion in relation to the chelatase-catalyzed reaction and on the biochemical and structural characterization of the ferrochelatase–frataxin partnership will be required to reveal how porphyrin distortion modulates the metal ion specificity of chelatases and how protein–protein interactions control the delivery of Fe2+ from frataxin to ferrochelatase.
We thank Tobias Karlberg for Figure 1. We apologize to investigators whose work could not be cited owing to space limitations. This work was supported by grants from the Swedish Research Council (to S. A.-K. and M. H.), the Muscular Dystrophy Association and National Institutes of Health (AG15709 to G. I.) and the American Cancer Society (RSG-96–05106-TBE to G.C.F). Sandia is a multiprogram laboratory operated by Sandia Corporation, a Lockheed Martin Company, for the United States Department of Energy under Contract DE-AC04–94AL85000 (J.A.S.).