Ferrochelatase at the millennium: structures, mechanisms and [2Fe-2S] clusters

Woodruff

2005

Biochemical Journal

The initial and the terminal three enzymes of the mammalian haem biosynthetic pathway are nuclear encoded, cytoplasmically synthesized and post-translationally translocated into the mitochondrion. The first enzyme, ALAS (5-aminolaevulinate synthase), occurs as an isoenzyme encoded on different chromosomes and is synthesized either as a housekeeping protein (ALAS-1) in all nonerythroid cell types, or only in differentiating erythroid precursor cells (ALAS-2). Both ALAS proteins possess mitochondrial targeting sequences that have putative haem-binding motifs. In the present study, evidence is presented demonstrating that two haembinding motifs in the leader sequence, as well as one present in the N-terminus of the mature ALAS-1 function in vivo in the haemregulated translocation of ALAS-1. Coproporphyrinogen oxidase, the antepenultimate pathway enzyme, possesses a leader sequence that is approx. 120 residues long. In contrast with an earlier report suggesting that only 30 residues were required for translocation of the coproporphyrinogen oxidase, we report that the complete leader is necessary for translocation and that this process is not haem-sensitive in vivo. PPO (protoporphyrinogen oxidase) lacks a typical mitochondrial targeting leader sequence and was found to be effectively targeted by just 17 N-terminal residues. Bacillus subtilis PPO, which is very similar to human PPO at its N-terminal end, is not targeted to the mitochondrion when expressed in mammalian cells, demonstrating that the translocation is highly specific with regard to both the length and spacing of charged residues in this targeting region. Ferrochelatase, the terminal enzyme, possesses a typical N-terminal leader sequence and no evidence of a role for the C-terminus was found in mitochondrial targeting.

Section: Introductionmentioning

confidence: 99%

Examination of mitochondrial protein targeting of haem synthetic enzymes: in vivo identification of three functional haem-responsive motifs in 5-aminolaevulinate synthase

Woodruff

2005

Biochemical Journal

Journal of Biological Chemistry

“…Ferrochelatase has been identified in a large number of organisms (reviewed in Refs. [1][2][3]. In eukaryotes, ferrochelatase is nucleus-encoded with an average of 400 -500 amino acid residues and associated with the inner mitochondrial membrane.…”

mentioning

confidence: 99%

“…Prokaryotic ferrochelatases are smaller, and whereas most are associated with the plasma membrane, some are soluble (reviewed in Ref. 3). [2Fe-2S] clusters have been identified in animal ferrochelatases (4,5), in the yeast Schizosaccharomyces pombe (6), and in the bacterial species Caulobacter crescentus and Mycobacterium tuberculosis (7), but their roles remain elusive.…”

mentioning

confidence: 99%

Probing the Active Site Loop Motif of Murine Ferrochelatase by Random Mutagenesis

Shi

Ferreira

2004

Ferrochelatase catalyzes the terminal step of the heme biosynthetic pathway by inserting ferrous iron into protoporphyrin IX. A conserved loop motif was shown to form part of the active site and contact the bound porphyrin by molecular dynamics calculations and structural analysis. We applied a random mutagenesis approach and steady-state kinetic analysis to assess the role of the loop motif in murine ferrochelatase function, particularly with respect to porphyrin interaction.

Proc. Natl. Acad. Sci. U.S.A.

“…The enzyme ferrochelatase catalyzes the insertion of Fe 2ϩ into protoporphyrin IX as the last step in heme biosynthesis pathway (11). It was proposed that the enzyme catalyzes the porphyrin metallation reaction by distorting the porphyrin substrate toward a transition state-like geometry in which the pyrrole nitrogen lone pairs are exposed for metal chelation (12).…”

mentioning

confidence: 99%

Structural evidence for substrate strain in antibody catalysis

Yin

Andryski

Beuscher

et al. 2003

The crystal structure of the Michaelis complex between the Fab fragment of ferrochelatase antibody 7G12 and its substrate mesoporphyrin has been solved to 2.6-Å resolution. The antibodybound mesoporphyrin clearly adopts a nonplanar conformation and reveals that the antibody catalyzes the porphyrin metallation reaction by straining͞distorting the bound substrate toward the transition-state configuration. The crystal structures of the Fab fragment of the germ-line precursor antibody to 7G12 and its complex with the hapten N-methylmesoporphyrin have also been solved. A comparison of these structures with the corresponding structures of the affinity-matured antibody 7G12 reveals the molecular mechanism by which the immune system evolves binding energy to catalyze this reaction.M odern theories of biological catalysis date from Haldane's theory of strain: ''using Fischer's lock and key simile, the key does not fit the lock perfectly but exercises a certain strain on it'' (1). Although the notion that enzymes use binding energy to strain or distort substrates is a fundamental theory of enzyme catalysis (2), it has proven difficult to experimentally validate (3). One approach that has proven effective in testing theories of biological catalysis involves the use of the programmable nature of antibody binding energy to evolve selective catalysts. Antibody catalysis has allowed us to dissect the energetic contributions of transition state stabilization, general base and covalent catalysis, and proximity effects to catalysis (4-9, 29). More recently, we showed that an antibody (7G12) raised against a strained ground-state mimic acted as an efficient porphyrin metallation catalyst (10). We now report the x-ray crystal structure of the Michaelis complex formed between the Fab fragment of the ferrochelatase antibody 7G12 and its substrate mesoporphyrin IX (MP), in which the bound substrate is distorted toward the transition-state conformation for metal insertion. The structure of this complex provides unequivocal structural evidence for the strain theory proposed by Haldane more than 70 years ago. Moreover, the detailed structural and biophysical characterization of the germ-line and affinity-matured antibodies has provided a detailed mechanistic picture of the immunological evolution of this strain mechanism.The enzyme ferrochelatase catalyzes the insertion of Fe 2ϩ into protoporphyrin IX as the last step in heme biosynthesis pathway (11). It was proposed that the enzyme catalyzes the porphyrin metallation reaction by distorting the porphyrin substrate toward a transition state-like geometry in which the pyrrole nitrogen lone pairs are exposed for metal chelation (12). To test this notion, antibody 7G12 was generated against N-methylmesoporphyrin (NMP) in which the porphyrin macrocycle is distorted due to alkylation at one of the pyrrole nitrogens. The resulting antibody was found to catalyze the metallation of MP by Zn 2ϩ with a catalytic efficiency comparable to that of the natural enzyme (10). The same antibody also catal...