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Mats Hansson - One of the best experts on this subject based on the ideXlab platform.
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Bacterial Ferrochelatase turns human: Tyr13 determines the apparent metal specificity of Bacillus subtilis Ferrochelatase
JBIC Journal of Biological Inorganic Chemistry, 2011Co-Authors: Mattias D. Hansson, Tobias Karlberg, Salam Al-karadaghi, Martin J. Warren, Stephen E J Rigby, Christopher A. G. Söderberg, Sreekanth Rajan, Mats HanssonAbstract:Ferrochelatase catalyzes the insertion of Fe^2+ into protoporphyrin IX. The enzymatic product heme (protoheme IX) is a well-known cofactor in a wide range of proteins. The insertion of metal ions other than Fe^2+ occurs rarely in vivo, but all Ferrochelatases that have been studied can insert Zn^2+ at a good rate in vitro. Co^2+, but not Cu^2+, is known to be a good substrate of the mammalian and Saccharomyces cerevisiae Ferrochelatases. In contrast, Cu^2+, but not Co^2+, has been found to be a good substrate of bacterial Bacillus subtilis Ferrochelatase. It is not known how Ferrochelatase discriminates between different metal ion substrates. Structural analysis of B. subtilis Ferrochelatase has shown that Tyr13 is an indirect ligand of Fe^2+ and a direct ligand of a copper mesoporphyrin product. A structure-based comparison revealed that Tyr13 aligns with a Met residue in the S. cerevisiae and human Ferrochelatases. Tyr13 was changed to Met in the B. subtilis enzyme by site-directed mutagenesis. Enzymatic measurements showed that the modified enzyme inserted Co^2+ at a higher rate than the wild-type B. subtilis Ferrochelatase, but it had lost the ability to use Cu^2+ as a substrate. Thus, the B. subtilis Tyr13Met Ferrochelatase showed the same metal specificity as that of the Ferrochelatases from S. cerevisiae and human.
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porphyrin binding and distortion and substrate specificity in the Ferrochelatase reaction the role of active site residues
Journal of Molecular Biology, 2008Co-Authors: T Karlberg, Gloria C. Ferreira, Mats Hansson, Mattias Hansson, Raymond K Yengo, Renzo Johansson, Hege O Thorvaldsen, Salam AlkaradaghiAbstract:The specific insertion of a divalent metal ion into tetrapyrrole macrocycles is catalyzed by a group of enzymes called chelatases. Distortion of the tetrapyrrole has been proposed to be an important component of the mechanism of metallation. We present the structures of two different inhibitor complexes: (1) N-methylmesoporphyrin (N-MeMP) with the His183Ala variant of Bacillus subtilis Ferrochelatase; (2) the wild-type form of the same enzyme with deuteroporphyrin IX 2,4-disulfonic acid dihydrochloride (dSDP). Analysis of the structures showed that only one N-MeMP isomer out of the eight possible was bound to the protein and it was different from the isomer that was earlier found to bind to the wild-type enzyme. A comparison of the distortion of this porphyrin with other porphyrin complexes of Ferrochelatase and a catalytic antibody with Ferrochelatase activity using normal-coordinate structural decomposition reveals that certain types of distortion are predominant in all these complexes. On the other hand, dSDP, which binds closer to the protein surface compared to N-MeMP, does not undergo any distortion upon binding to the protein, underscoring that the position of the porphyrin within the active site pocket is crucial for generating the distortion required for metal insertion. In addition, in contrast to the wild-type enzyme, Cu(2+)-soaking of the His183Ala variant complex did not show any traces of porphyrin metallation. Collectively, these results provide new insights into the role of the active site residues of Ferrochelatase in controlling stereospecificity, distortion and metallation.
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amino acid residues his183 and glu264 in bacillus subtilis Ferrochelatase direct and facilitate the insertion of metal ion into protoporphyrin ix
Biochemistry, 2007Co-Authors: Mattias Hansson, Salam Alkaradaghi, T Karlberg, Muhammad Arys Rahardja, Mats HanssonAbstract:Ferrochelatase catalyzes the terminal step in the heme biosynthetic pathway, i.e., the incorporation of Fe(II) into protoporphyrin IX. Various biochemical and biophysical methods have been used to probe the enzyme for metal binding residues and the location of the active site. However, the location of the metal binding site and the path of the metal into the porphyrin are still disputed. Using site-directed mutagenesis on Bacillus subtilis Ferrochelatase we demonstrate that exchange of the conserved residues His183 and Glu264 affects the metal affinity of the enzyme. We also present the first X-ray crystal structure of Ferrochelatase with iron. Only a single iron was found in the active site, coordinated in a square pyramidal fashion by two amino acid residues, His183 and Glu264, and three water molecules. This iron was not present in the structure of a His183Ala modified Ferrochelatase. The results strongly suggest that the insertion of a metal ion into protoporphyrin IX by Ferrochelatase occurs from a metal binding site represented by His183 and Glu264.
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Metallation of the transition-state inhibitor N-methyl mesoporphyrin by Ferrochelatase: implications for the catalytic reaction mechanism.
Journal of molecular biology, 2005Co-Authors: Stepan Shipovskov, Tobias Karlberg, Michel Fodje, Mattias D. Hansson, Gloria C. Ferreira, Mats Hansson, Curt T. Reimann, Salam Al-karadaghiAbstract:Insertion of metals into various tetrapyrroles is catalysed by a group of enzymes called chelatases, e.g. nickel, cobalt, magnesium and ferro-chelatase. It has been proposed that catalytic metallation includes distorting the porphyrin substrate by the enzyme towards a transition state-like geometry in which at least one of the pyrrole rings will be available for metal chelation. Here, we present a study of metal insertion into the transition-state inhibitor of protoporphyrin IX Ferrochelatase, N-methyl mesoporphyrin (N-MeMP), by time-resolved crystallography and mass spectrometry with and without the presence of Ferrochelatase. The results show that metallation of N-MeMP has a very limited effect on the conformation of the residues that participate in porphyrin and metal binding. These findings support theoretical data, which indicate that product release is controlled largely by the strain created by metal insertion into the distorted porphyrin. The results suggest that, similar to non-catalytic metallation of N-MeMP, the Ferrochelatase-assisted metallation depends on the ligand exchange rate for the respective metal. Moreover, Ferrochelatase catalyses insertion of Cu(II) and Zn(II) into N-MeMP with a rate that is about 20 times faster than non-enzymatic metallation in solution, suggesting that the catalytic strategy of Ferrochelatase includes a stage of acceleration of the rate of ligand exchange for the metal substrate. The greater efficiency of N-MeMP metallation by Cu(II), as compared to Zn(II), contrasts with the K(m) values for Zn(II) (17 microM) and Cu(II) (170 microM) obtained for metallation of protoporphyrin IX. We suggest that this difference in metal specificity depends on the type of distortion imposed by the enzyme on protoporphyrin IX, which is different from the intrinsic non-planar distortion of N-MeMP. A mechanism of control of metal specificity by porphyrin distortion may be general for different chelatases, and may have common features with the mechanism of metal specificity in crown ethers.
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metal binding to bacillus subtilis Ferrochelatase and interaction between metal sites
Journal of Biological Inorganic Chemistry, 2003Co-Authors: D. Lecerof, Michel Fodje, Mats Hansson, Ulf Olsson, Andreas Hansson, Emma Sigfridsson, Ulf Ryde, Roman Alvarez Leon, Salam AlkaradaghiAbstract:Ferrochelatase, the terminal enzyme in heme biosynthesis, catalyses metal insertion into protoporphyrin IX. The location of the metal binding site with respect to the bound porphyrin substrate and the mode of metal binding are of central importance for understanding the mechanism of porphyrin metallation. In this work we demonstrate that Zn2+, which is commonly used as substrate in assays of the Ferrochelatase reaction, and Cd2+, an inhibitor of the enzyme, bind to the invariant amino acids His183 and Glu264 and water molecules, all located within the porphyrin binding cleft. On the other hand, Mg2+, which has been shown to bind close to the surface at 7 A from His183, was largely absent from its site. Activity measurements demonstrate that Mg2+ has a stimulatory effect on the enzyme, lowering KM for Zn2+ from 55 to 24 µM. Changing one of the Mg2+ binding residues, Glu272, to serine abolishes the effect of Mg2+. It is proposed that prior to metal insertion the metal may form a sitting-atop (SAT) complex with the invariant His-Glu couple and the porphyrin. Metal binding to the Mg2+ site may stimulate metal release from the protein ligands and its insertion into the porphyrin.
Salam Alkaradaghi - One of the best experts on this subject based on the ideXlab platform.
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the structure of the complex between yeast frataxin and Ferrochelatase characterization and pre steady state reaction of ferrous iron delivery and heme synthesis
Journal of Biological Chemistry, 2016Co-Authors: Christopher G Soderberg, Gloria C. Ferreira, Mallory E Gillam, Evachristina Ahlgren, Gregory A Hunter, Oleksandr Gakh, Grazia Isaya, Salam AlkaradaghiAbstract:Frataxin is a mitochondrial iron-binding protein involved in iron storage, detoxification, and delivery for iron sulfur-cluster assembly and heme biosynthesis. The ability of frataxin from different organisms to populate multiple oligomeric states in the presence of metal ions, e.g. Fe(2+) and Co(2+), led to the suggestion that different oligomers contribute to the functions of frataxin. Here we report on the complex between yeast frataxin and Ferrochelatase, the terminal enzyme of heme biosynthesis. Protein-protein docking and cross-linking in combination with mass spectroscopic analysis and single-particle reconstruction from negatively stained electron microscopic images were used to verify the Yfh1-Ferrochelatase interactions. The model of the complex indicates that at the 2:1 Fe(2+)-to-protein ratio, when Yfh1 populates a trimeric state, there are two interaction interfaces between frataxin and the Ferrochelatase dimer. Each interaction site involves one Ferrochelatase monomer and one frataxin trimer, with conserved polar and charged amino acids of the two proteins positioned at hydrogen-bonding distances from each other. One of the subunits of the Yfh1 trimer interacts extensively with one subunit of the Ferrochelatase dimer, contributing to the stability of the complex, whereas another trimer subunit is positioned for Fe(2+) delivery. Single-turnover stopped-flow kinetics experiments demonstrate that increased rates of heme production result from monomers, dimers, and trimers, indicating that these forms are most efficient in delivering Fe(2+) to Ferrochelatase and sustaining porphyrin metalation. Furthermore, they support the proposal that frataxin-mediated delivery of this potentially toxic substrate overcomes formation of reactive oxygen species.
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porphyrin binding and distortion and substrate specificity in the Ferrochelatase reaction the role of active site residues
Journal of Molecular Biology, 2008Co-Authors: T Karlberg, Gloria C. Ferreira, Mats Hansson, Mattias Hansson, Raymond K Yengo, Renzo Johansson, Hege O Thorvaldsen, Salam AlkaradaghiAbstract:The specific insertion of a divalent metal ion into tetrapyrrole macrocycles is catalyzed by a group of enzymes called chelatases. Distortion of the tetrapyrrole has been proposed to be an important component of the mechanism of metallation. We present the structures of two different inhibitor complexes: (1) N-methylmesoporphyrin (N-MeMP) with the His183Ala variant of Bacillus subtilis Ferrochelatase; (2) the wild-type form of the same enzyme with deuteroporphyrin IX 2,4-disulfonic acid dihydrochloride (dSDP). Analysis of the structures showed that only one N-MeMP isomer out of the eight possible was bound to the protein and it was different from the isomer that was earlier found to bind to the wild-type enzyme. A comparison of the distortion of this porphyrin with other porphyrin complexes of Ferrochelatase and a catalytic antibody with Ferrochelatase activity using normal-coordinate structural decomposition reveals that certain types of distortion are predominant in all these complexes. On the other hand, dSDP, which binds closer to the protein surface compared to N-MeMP, does not undergo any distortion upon binding to the protein, underscoring that the position of the porphyrin within the active site pocket is crucial for generating the distortion required for metal insertion. In addition, in contrast to the wild-type enzyme, Cu(2+)-soaking of the His183Ala variant complex did not show any traces of porphyrin metallation. Collectively, these results provide new insights into the role of the active site residues of Ferrochelatase in controlling stereospecificity, distortion and metallation.
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amino acid residues his183 and glu264 in bacillus subtilis Ferrochelatase direct and facilitate the insertion of metal ion into protoporphyrin ix
Biochemistry, 2007Co-Authors: Mattias Hansson, Salam Alkaradaghi, T Karlberg, Muhammad Arys Rahardja, Mats HanssonAbstract:Ferrochelatase catalyzes the terminal step in the heme biosynthetic pathway, i.e., the incorporation of Fe(II) into protoporphyrin IX. Various biochemical and biophysical methods have been used to probe the enzyme for metal binding residues and the location of the active site. However, the location of the metal binding site and the path of the metal into the porphyrin are still disputed. Using site-directed mutagenesis on Bacillus subtilis Ferrochelatase we demonstrate that exchange of the conserved residues His183 and Glu264 affects the metal affinity of the enzyme. We also present the first X-ray crystal structure of Ferrochelatase with iron. Only a single iron was found in the active site, coordinated in a square pyramidal fashion by two amino acid residues, His183 and Glu264, and three water molecules. This iron was not present in the structure of a His183Ala modified Ferrochelatase. The results strongly suggest that the insertion of a metal ion into protoporphyrin IX by Ferrochelatase occurs from a metal binding site represented by His183 and Glu264.
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metal binding to bacillus subtilis Ferrochelatase and interaction between metal sites
Journal of Biological Inorganic Chemistry, 2003Co-Authors: D. Lecerof, Michel Fodje, Mats Hansson, Ulf Olsson, Andreas Hansson, Emma Sigfridsson, Ulf Ryde, Roman Alvarez Leon, Salam AlkaradaghiAbstract:Ferrochelatase, the terminal enzyme in heme biosynthesis, catalyses metal insertion into protoporphyrin IX. The location of the metal binding site with respect to the bound porphyrin substrate and the mode of metal binding are of central importance for understanding the mechanism of porphyrin metallation. In this work we demonstrate that Zn2+, which is commonly used as substrate in assays of the Ferrochelatase reaction, and Cd2+, an inhibitor of the enzyme, bind to the invariant amino acids His183 and Glu264 and water molecules, all located within the porphyrin binding cleft. On the other hand, Mg2+, which has been shown to bind close to the surface at 7 A from His183, was largely absent from its site. Activity measurements demonstrate that Mg2+ has a stimulatory effect on the enzyme, lowering KM for Zn2+ from 55 to 24 µM. Changing one of the Mg2+ binding residues, Glu272, to serine abolishes the effect of Mg2+. It is proposed that prior to metal insertion the metal may form a sitting-atop (SAT) complex with the invariant His-Glu couple and the porphyrin. Metal binding to the Mg2+ site may stimulate metal release from the protein ligands and its insertion into the porphyrin.
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metal binding to saccharomyces cerevisiae Ferrochelatase
Biochemistry, 2002Co-Authors: T Karlberg, Mats Hansson, D. Lecerof, Monika Gora, Germund Silvegren, Rosine Labbebois, Salam AlkaradaghiAbstract:Ferrochelatase is the terminal enzyme in the heme biosynthetic pathway. It catalyzes the insertion of ferrous iron into protoporphyrin IX to produce protoheme IX. The crystal structures of Ferrochelatase from Saccharomyces cerevisiae in free form, in complex with Co(II), a substrate metal ion, and in complex with two inhibitors, Cd(II) and Hg(I), are presented in this work. The enzyme is a homodimer, with clear asymmetry between the monomers with regard to the porphyrin binding cleft and the mode of metal binding. The Co(II) and Cd(II) complexes reveal the metal binding site which consists of the invariant amino acids H235, E314, and S275 and solvent molecules. The shortest distance to the metal reveals that amino acid H235 is the primary metal binding residue. A second site with bound Cd(II) was found close to the surface of the molecule, approximately 14 A from H235, with E97, H317, and E326 participating in metal coordination. It is suggested that this site corresponds to the magnesium binding site in Bacillus subtilis Ferrochelatase. The latter site is also located at the surface of the molecule and thought to be involved in initial metal binding and regulation.
Martin J. Warren - One of the best experts on this subject based on the ideXlab platform.
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2014b) Identification and characterization of the ‘missing’ terminal enzyme for siroheme biosynthesis in alpha-proteobacteria. Mol Microbiol 92
2016Co-Authors: Shilpa Bali, Sarah E Rollauer, Evelyne Raux-deery, Pietro Roversi, Martin J. Warren, Susan M. Lea, Stuart J FergusonAbstract:It has recently been shown that the biosynthetic route for both the d1-haem cofactor of dissimilatory cd1 nitrite reductases and haem, via the novel alternative-haem-synthesis pathway, involves siroheme as an intermediate, which was previously thought to occur only as a cofactor in assimilatory sulphite/ nitrite reductases. In many denitrifiers (which require d1-haem), the pathway to make siroheme remained to be identified. Here we identify and characterize a sirohydrochlorin–Ferrochelatase from Paracoccus pantotrophus that catalyses the last step of siroheme synthesis. It is encoded by a gene annotated as cbiX that was previously assumed to be encoding a cobal-tochelatase, acting on sirohydrochlorin. Expressing this chelatase from a plasmid restored the wild-type phenotype of an Escherichia coli mutant-strain lacking sirohydrochlorin–Ferrochelatase activity, showing that this chelatase can act in the in vivo siroheme synthesis. A ΔcbiX mutant in P. denitrificans was unable to respire anaerobically on nitrate, proving the role of siroheme as a precursor to another cofactor. We report the 1.9 Å crystal structure of this Ferrochelatase. In vivo analysis of single amino acid variants of this chelatase suggests that two histidines, His127 and His187, are essential for siroheme synthesis. This CbiX can generally be identified in α-proteobacteria as the terminal enzyme of siroheme biosynthesis
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identification and characterization of the missing terminal enzyme for siroheme biosynthesis in α proteobacteria
Molecular Microbiology, 2014Co-Authors: Shilpa Bali, Sarah E Rollauer, Evelyne Rauxdeery, Pietro Roversi, Martin J. Warren, Stuart J FergusonAbstract:It has recently been shown that the biosynthetic route for both the d1-haem cofactor of dissimilatory cd1 nitrite reductases and haem, via the novel alternative-haem-synthesis pathway, involves siroheme as an intermediate, which was previously thought to occur only as a cofactor in assimilatory sulphite/nitrite reductases. In many denitrifiers (which require d1-haem), the pathway to make siroheme remained to be identified. Here we identify and characterize a sirohydrochlorin–Ferrochelatase from Paracoccus pantotrophus that catalyses the last step of siroheme synthesis. It is encoded by a gene annotated as cbiX that was previously assumed to be encoding a cobaltochelatase, acting on sirohydrochlorin. Expressing this chelatase from a plasmid restored the wild-type phenotype of an Escherichia coli mutant-strain lacking sirohydrochlorin–Ferrochelatase activity, showing that this chelatase can act in the in vivo siroheme synthesis. A ΔcbiX mutant in P. denitrificans was unable to respire anaerobically on nitrate, proving the role of siroheme as a precursor to another cofactor. We report the 1.9 A crystal structure of this Ferrochelatase. In vivo analysis of single amino acid variants of this chelatase suggests that two histidines, His127 and His187, are essential for siroheme synthesis. This CbiX can generally be identified in α-proteobacteria as the terminal enzyme of siroheme biosynthesis.
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Bacterial Ferrochelatase turns human: Tyr13 determines the apparent metal specificity of Bacillus subtilis Ferrochelatase
JBIC Journal of Biological Inorganic Chemistry, 2011Co-Authors: Mattias D. Hansson, Tobias Karlberg, Salam Al-karadaghi, Martin J. Warren, Stephen E J Rigby, Christopher A. G. Söderberg, Sreekanth Rajan, Mats HanssonAbstract:Ferrochelatase catalyzes the insertion of Fe^2+ into protoporphyrin IX. The enzymatic product heme (protoheme IX) is a well-known cofactor in a wide range of proteins. The insertion of metal ions other than Fe^2+ occurs rarely in vivo, but all Ferrochelatases that have been studied can insert Zn^2+ at a good rate in vitro. Co^2+, but not Cu^2+, is known to be a good substrate of the mammalian and Saccharomyces cerevisiae Ferrochelatases. In contrast, Cu^2+, but not Co^2+, has been found to be a good substrate of bacterial Bacillus subtilis Ferrochelatase. It is not known how Ferrochelatase discriminates between different metal ion substrates. Structural analysis of B. subtilis Ferrochelatase has shown that Tyr13 is an indirect ligand of Fe^2+ and a direct ligand of a copper mesoporphyrin product. A structure-based comparison revealed that Tyr13 aligns with a Met residue in the S. cerevisiae and human Ferrochelatases. Tyr13 was changed to Met in the B. subtilis enzyme by site-directed mutagenesis. Enzymatic measurements showed that the modified enzyme inserted Co^2+ at a higher rate than the wild-type B. subtilis Ferrochelatase, but it had lost the ability to use Cu^2+ as a substrate. Thus, the B. subtilis Tyr13Met Ferrochelatase showed the same metal specificity as that of the Ferrochelatases from S. cerevisiae and human.
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identification and characterization of the terminal enzyme of siroheme biosynthesis from arabidopsis thaliana a plastid located sirohydrochlorin Ferrochelatase containing a 2fe 2s center
Journal of Biological Chemistry, 2005Co-Authors: Evelyne Rauxdeery, Helen K Leech, Kerry Ann Nakrieko, Alison G. Smith, Kirsty J Mclean, Andrew W Munro, Peter Heathcote, Stephen E J Rigby, Martin J. WarrenAbstract:Abstract Higher plant sulfite and nitrite reductases contain siroheme as a prosthetic group. Siroheme is synthesized from the tetrapyrrole primogenitor uroporphyrinogen III in three steps involving methylation, oxidation, and ferrochelation reactions. In this paper we report on the Arabidopsis thaliana sirohydrochlorin Ferrochelatase At-SirB. The complete precursor protein of 225 amino acids and shorter constructs in which the first 46 or 79 residues had been removed were shown to complement a defined Escherichia coli sirohydrochlorin Ferrochelatase mutant. The mature form of the protein appeared to consist of only 150 amino acids, making it much smaller than previously characterized Ferrochelatases. Green fluorescent protein tagging revealed that it is located in the chloroplast. The enzyme was easily produced in E. coli as a recombinant protein, and the isolated enzyme was found to have a specific activity of 48.5 nmol/min/mg. Significantly, the protein purified as a brown-colored solution with a UV-visible spectrum containing maxima at 415 and 455 nm, suggestive of an Fe-S center. EPR analysis of the recombinant protein produced a rhombic spectrum with G-values of 2.04, 1.94, and 1.90 and with temperature dependence consistent with a 2Fe-2S center. Redox titration demonstrated that the Fe-S center is highly unstable, with an apparent midpoint reduction potential of about -370 mV. This is the first Fe-S center to be reported in a higher plant Ferrochelatase. The implications of the Fe-S center in an enzyme that is so closely associated with the metabolism of sulfur and iron are discussed.
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common chelatase design in the branched tetrapyrrole pathways of heme and anaerobic cobalamin synthesis
Biochemistry, 1999Co-Authors: Heidi L Schubert, Evelyne Raux, Keith S Wilson, Martin J. WarrenAbstract:Prosthetic groups such as heme, chlorophyll, and cobalamin (vitamin B(12)) are characterized by their branched biosynthetic pathway and unique metal insertion steps. The metal ion chelatases can be broadly classed either as single-subunit ATP-independent enzymes, such as the anaerobic cobalt chelatase and the protoporphyrin IX (PPIX) Ferrochelatase, or as heterotrimeric, ATP-dependent enzymes, such as the Mg chelatase involved in chlorophyll biosynthesis. The X-ray structure of the anaerobic cobalt chelatase from Salmonella typhimurium, CbiK, has been solved to 2.4 A resolution. Despite a lack of significant amino acid sequence similarity, the protein structure is homologous to that of Bacillus subtilis PPIX Ferrochelatase. Both enzymes contain a histidine residue previously identified as the metal ion ligand, but CbiK contains a second histidine in place of the glutamic acid residue identified as a general base in PPIX Ferrochelatase. Site-directed mutagenesis has confirmed a role for this histidine and a nearby glutamic acid in cobalt binding, modulating metal ion specificity as well as catalytic efficiency. Contrary to the predicted protoporphyrin binding site in PPIX Ferrochelatase, the precorrin-2 binding site in CbiK is clearly defined within a large horizontal cleft between the N- and C-terminal domains. The structural similarity has implications for the understanding of the evolution of this branched biosynthetic pathway.
Gloria C. Ferreira - One of the best experts on this subject based on the ideXlab platform.
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Ferrochelatase π helix implications from examining the role of the conserved π helix glutamates in porphyrin metalation and product release
Archives of Biochemistry and Biophysics, 2018Co-Authors: Mallory E Gillam, Gregory A Hunter, Gloria C. FerreiraAbstract:Abstract Protoporphyrin Ferrochelatase catalyzes the insertion of Fe2+ into protoporphyrin IX to form heme. To determine whether a conserved, active site π-helix contributes to the translocation of the metal ion substrate to the Ferrochelatase-bound porphyrin substrate, the invariant π-helix glutamates were replaced with amino acids with non-negatively charged side chains, and the kinetic mechanisms of the generated variants were examined. Analysis of yeast wild-type Ferrochelatase-, E314Q- and E318Q-catalyzed reactions, under multi- and single-turnover conditions, demonstrated that the mutations of the π-helix glutamates hindered both protoporphyrin metalation and release of the metalated porphyrin, by slowing each step by approximately 30–50%. Protoporphyrin metalation occurred with an apparent pKa of 7.3 ± 0.1, which was assigned to binding of Fe2+ by deprotonated Glu-314 and Glu-314-assisted Fe2+ insertion into the porphyrin ring. We propose that unwinding of the π-helix concomitant with the adoption of a protein open conformation positions the deprotonated Glu-314 to bind Fe2+ from the surface of the enzyme. Transition to the closed conformation, with π-helix winding, brings Glu-314-bound Fe2+ to the active site for incorporation into protoporphyrin.
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the structure of the complex between yeast frataxin and Ferrochelatase characterization and pre steady state reaction of ferrous iron delivery and heme synthesis
Journal of Biological Chemistry, 2016Co-Authors: Christopher G Soderberg, Gloria C. Ferreira, Mallory E Gillam, Evachristina Ahlgren, Gregory A Hunter, Oleksandr Gakh, Grazia Isaya, Salam AlkaradaghiAbstract:Frataxin is a mitochondrial iron-binding protein involved in iron storage, detoxification, and delivery for iron sulfur-cluster assembly and heme biosynthesis. The ability of frataxin from different organisms to populate multiple oligomeric states in the presence of metal ions, e.g. Fe(2+) and Co(2+), led to the suggestion that different oligomers contribute to the functions of frataxin. Here we report on the complex between yeast frataxin and Ferrochelatase, the terminal enzyme of heme biosynthesis. Protein-protein docking and cross-linking in combination with mass spectroscopic analysis and single-particle reconstruction from negatively stained electron microscopic images were used to verify the Yfh1-Ferrochelatase interactions. The model of the complex indicates that at the 2:1 Fe(2+)-to-protein ratio, when Yfh1 populates a trimeric state, there are two interaction interfaces between frataxin and the Ferrochelatase dimer. Each interaction site involves one Ferrochelatase monomer and one frataxin trimer, with conserved polar and charged amino acids of the two proteins positioned at hydrogen-bonding distances from each other. One of the subunits of the Yfh1 trimer interacts extensively with one subunit of the Ferrochelatase dimer, contributing to the stability of the complex, whereas another trimer subunit is positioned for Fe(2+) delivery. Single-turnover stopped-flow kinetics experiments demonstrate that increased rates of heme production result from monomers, dimers, and trimers, indicating that these forms are most efficient in delivering Fe(2+) to Ferrochelatase and sustaining porphyrin metalation. Furthermore, they support the proposal that frataxin-mediated delivery of this potentially toxic substrate overcomes formation of reactive oxygen species.
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metal ion substrate inhibition of Ferrochelatase
Journal of Biological Chemistry, 2008Co-Authors: Gregory A Hunter, Matthew Sampson, Gloria C. FerreiraAbstract:Ferrochelatase catalyzes the insertion of ferrous iron into protoporphyrin IX to form heme. Robust kinetic analyses of the reaction mechanism are complicated by the instability of ferrous iron in aqueous solution, particularly at alkaline pH values. At pH 7.00 the half-life for spontaneous oxidation of ferrous ion is approximately 2 min in the absence of metal complexing additives, which is sufficient for direct comparisons of alternative metal ion substrates with iron. These analyses reveal that purified recombinant Ferrochelatase from both murine and yeast sources inserts not only ferrous iron but also divalent cobalt, zinc, nickel, and copper into protoporphyrin IX to form the corresponding metalloporphyrins but with considerable mechanistic variability. Ferrous iron is the preferred metal ion substrate in terms of apparent kcat and is also the only metal ion substrate not subject to severe substrate inhibition. Substrate inhibition occurs in the order Cu2+ > Zn2+ > Co2+ > Ni2+ and can be alleviated by the addition of metal complexing agents such as β-mercaptoethanol or imidazole to the reaction buffer. These data indicate the presence of two catalytically significant metal ion binding sites that may coordinately regulate a selective processivity for the various potential metal ion substrates.
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porphyrin binding and distortion and substrate specificity in the Ferrochelatase reaction the role of active site residues
Journal of Molecular Biology, 2008Co-Authors: T Karlberg, Gloria C. Ferreira, Mats Hansson, Mattias Hansson, Raymond K Yengo, Renzo Johansson, Hege O Thorvaldsen, Salam AlkaradaghiAbstract:The specific insertion of a divalent metal ion into tetrapyrrole macrocycles is catalyzed by a group of enzymes called chelatases. Distortion of the tetrapyrrole has been proposed to be an important component of the mechanism of metallation. We present the structures of two different inhibitor complexes: (1) N-methylmesoporphyrin (N-MeMP) with the His183Ala variant of Bacillus subtilis Ferrochelatase; (2) the wild-type form of the same enzyme with deuteroporphyrin IX 2,4-disulfonic acid dihydrochloride (dSDP). Analysis of the structures showed that only one N-MeMP isomer out of the eight possible was bound to the protein and it was different from the isomer that was earlier found to bind to the wild-type enzyme. A comparison of the distortion of this porphyrin with other porphyrin complexes of Ferrochelatase and a catalytic antibody with Ferrochelatase activity using normal-coordinate structural decomposition reveals that certain types of distortion are predominant in all these complexes. On the other hand, dSDP, which binds closer to the protein surface compared to N-MeMP, does not undergo any distortion upon binding to the protein, underscoring that the position of the porphyrin within the active site pocket is crucial for generating the distortion required for metal insertion. In addition, in contrast to the wild-type enzyme, Cu(2+)-soaking of the His183Ala variant complex did not show any traces of porphyrin metallation. Collectively, these results provide new insights into the role of the active site residues of Ferrochelatase in controlling stereospecificity, distortion and metallation.
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Metallation of the transition-state inhibitor N-methyl mesoporphyrin by Ferrochelatase: implications for the catalytic reaction mechanism.
Journal of molecular biology, 2005Co-Authors: Stepan Shipovskov, Tobias Karlberg, Michel Fodje, Mattias D. Hansson, Gloria C. Ferreira, Mats Hansson, Curt T. Reimann, Salam Al-karadaghiAbstract:Insertion of metals into various tetrapyrroles is catalysed by a group of enzymes called chelatases, e.g. nickel, cobalt, magnesium and ferro-chelatase. It has been proposed that catalytic metallation includes distorting the porphyrin substrate by the enzyme towards a transition state-like geometry in which at least one of the pyrrole rings will be available for metal chelation. Here, we present a study of metal insertion into the transition-state inhibitor of protoporphyrin IX Ferrochelatase, N-methyl mesoporphyrin (N-MeMP), by time-resolved crystallography and mass spectrometry with and without the presence of Ferrochelatase. The results show that metallation of N-MeMP has a very limited effect on the conformation of the residues that participate in porphyrin and metal binding. These findings support theoretical data, which indicate that product release is controlled largely by the strain created by metal insertion into the distorted porphyrin. The results suggest that, similar to non-catalytic metallation of N-MeMP, the Ferrochelatase-assisted metallation depends on the ligand exchange rate for the respective metal. Moreover, Ferrochelatase catalyses insertion of Cu(II) and Zn(II) into N-MeMP with a rate that is about 20 times faster than non-enzymatic metallation in solution, suggesting that the catalytic strategy of Ferrochelatase includes a stage of acceleration of the rate of ligand exchange for the metal substrate. The greater efficiency of N-MeMP metallation by Cu(II), as compared to Zn(II), contrasts with the K(m) values for Zn(II) (17 microM) and Cu(II) (170 microM) obtained for metallation of protoporphyrin IX. We suggest that this difference in metal specificity depends on the type of distortion imposed by the enzyme on protoporphyrin IX, which is different from the intrinsic non-planar distortion of N-MeMP. A mechanism of control of metal specificity by porphyrin distortion may be general for different chelatases, and may have common features with the mechanism of metal specificity in crown ethers.
Alison G. Smith - One of the best experts on this subject based on the ideXlab platform.
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identification and characterization of the terminal enzyme of siroheme biosynthesis from arabidopsis thaliana a plastid located sirohydrochlorin Ferrochelatase containing a 2fe 2s center
Journal of Biological Chemistry, 2005Co-Authors: Evelyne Rauxdeery, Helen K Leech, Kerry Ann Nakrieko, Alison G. Smith, Kirsty J Mclean, Andrew W Munro, Peter Heathcote, Stephen E J Rigby, Martin J. WarrenAbstract:Abstract Higher plant sulfite and nitrite reductases contain siroheme as a prosthetic group. Siroheme is synthesized from the tetrapyrrole primogenitor uroporphyrinogen III in three steps involving methylation, oxidation, and ferrochelation reactions. In this paper we report on the Arabidopsis thaliana sirohydrochlorin Ferrochelatase At-SirB. The complete precursor protein of 225 amino acids and shorter constructs in which the first 46 or 79 residues had been removed were shown to complement a defined Escherichia coli sirohydrochlorin Ferrochelatase mutant. The mature form of the protein appeared to consist of only 150 amino acids, making it much smaller than previously characterized Ferrochelatases. Green fluorescent protein tagging revealed that it is located in the chloroplast. The enzyme was easily produced in E. coli as a recombinant protein, and the isolated enzyme was found to have a specific activity of 48.5 nmol/min/mg. Significantly, the protein purified as a brown-colored solution with a UV-visible spectrum containing maxima at 415 and 455 nm, suggestive of an Fe-S center. EPR analysis of the recombinant protein produced a rhombic spectrum with G-values of 2.04, 1.94, and 1.90 and with temperature dependence consistent with a 2Fe-2S center. Redox titration demonstrated that the Fe-S center is highly unstable, with an apparent midpoint reduction potential of about -370 mV. This is the first Fe-S center to be reported in a higher plant Ferrochelatase. The implications of the Fe-S center in an enzyme that is so closely associated with the metabolism of sulfur and iron are discussed.
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expression analysis of the two Ferrochelatase genes in arabidopsis in different tissues and under stress conditions reveals their different roles in haem biosynthesis
Plant Molecular Biology, 2002Co-Authors: Davinder Pal Singh, Johanna E Cornah, Sophie Hadingham, Alison G. SmithAbstract:The Arabidopsis thaliana genome has two genes (AtFC-I and AtFC-II), encoding Ferrochelatase, the terminal enzyme of haem biosynthesis. The roles of the two enzymes in the synthesis of haem for different haemoproteins was investigated using reporter gene analysis. A 1.41 kb fragment from the 5' upstream region of the AtFC-II gene was fused to the luciferase gene, and then introduced into tobacco plants, followed by luciferase activity measurements. AtFC-II-LUCwas expressed in all aerial parts of the plant, and was highest in flowers, but it was not expressed in roots. It was unaffected by viral infection, and considerably reduced by wounding or oxidative stress. Similarly, a 1.76 kb region of the AtFC-I promoter was fused to the uidA gene encoding beta-glucuronidase. AtFC-I-GUS was expressed in all tissues of the plant, but was higher in roots and flowers than in leaves or stems. It was induced by sucrose, wounding and oxidative stress and, most markedly, by plants undergoing the hypersensitive response to TMV infection. Levels of endogenous Ferrochelatase activity were increased in pea chloroplasts isolated from wounded leaves, indicating that the induction in promoter activity is likely to result in increased haem biosynthetic potential. Salicylic acid, but not methyl-jasmonate was able to replace the stress treatment in induction of AtFC-I expression, suggesting that the requirement for haem synthesis is part of the defence response. The implications of the results for the different roles of the two Ferrochelatases in haem biosynthesis are discussed.
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measurement of Ferrochelatase activity using a novel assay suggests that plastids are the major site of haem biosynthesis in both photosynthetic and non photosynthetic cells of pea pisum sativum l
Biochemical Journal, 2002Co-Authors: Johanna E Cornah, Davinder Pal Singh, Jennifer M Roper, Alison G. SmithAbstract:Ferrochelatase is the terminal enzyme of haem biosynthesis, catalysing the insertion of ferrous iron into the macrocycle of protoporphyrin IX, the last common intermediate of haem and chlorophyll synthesis. Its activity has been reported in both plastids and mitochondria of higher plants, but the relative amounts of the enzyme in the two organelles are unknown. Ferrochelatase is difficult to assay since ferrous iron requires strict anaerobic conditions to prevent oxidation, and in photosynthetic tissues chlorophyll interferes with the quantification of the product. Accordingly, we developed a sensitive fluorimetric assay for Ferrochelatase that employs Co(2+) and deuteroporphyrin in place of the natural substrates, and measures the decrease in deuteroporphyrin fluorescence. A hexane-extraction step to remove chlorophyll is included for green tissue. The assay is linear over a range of chloroplast protein concentrations, with an average specific activity of 0.68 nmol x min(-1) x mg of protein(-1), the highest yet reported. The corresponding value for mitochondria is 0.19 nmol x min(-1) x mg of protein(-1). The enzyme is inhibited by N-methylprotoporphyrin, with an estimated IC(50) value of approximately 1 nM. Using this assay we have quantified Ferrochelatase activity in plastids and mitochondria from green pea leaves, etiolated pea leaves and pea roots to determine the relative amounts in the two organelles. We found that, in all three tissues, greater than 90% of the activity was associated with plastids, but Ferrochelatase was reproducibly detected in mitochondria, at levels greater than the contaminating plastid marker enzyme, and was latent. Our results indicate that plastids are the major site of haem biosynthesis in higher plant cells, but that mitochondria also have the capacity for haem production.
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two types of Ferrochelatase in photosynthetic and nonphotosynthetic tissues of cucumber their difference in phylogeny gene expression and localization
Journal of Biological Chemistry, 2002Co-Authors: Takuo Suzuki, Alison G. Smith, Tatsuru Masuda, Davinder Pal Singh, Fuiching Tan, Tohru Tsuchiya, Hiroshi Shimada, Hiroyuki Ohta, Kenichiro TakamiyaAbstract:Ferrochelatase catalyzes the insertion of Fe2+ into protoporphyrin IX to generate protoheme. In higher plants, there is evidence for two isoforms of this enzyme that fulfill different roles. Here, we describe the isolation of a second Ferrochelatase cDNA from cucumber (CsFeC2) that was less similar to a previously isolated isoform (CsFeC1) than it was to some Ferrochelatases from other higher plants. Inin vitro import experiments, the two cucumber isoforms showed characteristics similar to their respective Ferrochelatase counterparts of Arabidopsis thaliana. The C-terminal region of CsFeC2 but not CsFeC1 contained a conserved motif found in light-harvesting chlorophyll proteins, and CsFeC2 belonged to a phylogenetic group of plant Ferrochelatases containing this conserved motif. We demonstrate that CsFeC2 was localized predominantly in thylakoid membranes as an intrinsic protein, and forming complexes probably with the C-terminal conserved motif, but a minor portion was also detected in envelope membranes. CsFeC2 mRNA was detected in all tissues and was light-responsive in cotyledons, whereasCsFeC1 mRNA was detected in nonphotosynthetic tissues and was not light-responsive. Interestingly, tissue-, light-, and cycloheximide-dependent expressions of the two isoforms of Ferrochelatase were similar to those of two glutamyl-tRNA reductase isoforms involved in the early step of tetrapyrrole biosynthesis, suggesting the existence of distinctly controlled tetrapyrrole biosynthetic pathways in photosynthetic and nonphotosynthetic tissues.
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two different genes encode Ferrochelatase in arabidopsis mapping expression and subcellular targeting of the precursor proteins
Plant Journal, 1998Co-Authors: Keng See Chow, Davinder Pal Singh, Amanda R Walker, Alison G. SmithAbstract:Summary Ferrochelatase is the last enzyme of haem biosynthesis. We have isolated 27 independent Ferrochelatase cDNAs fromArabidopsis thalianaby functional complementation of a yeast mutant. Twenty-two of these cDNAs were similar to a previously isolated clone, AF3, and although they varied in length at the 5′ and 3′ ends, their nucleotide sequences were identical, indicating that they were derived from the same gene (Ferrochelatase-I). The remaining five cDNAs all encoded a separate Ferrochelatase isoform (Ferrochelatase-II), which was 69% identical at the amino acid level to Ferrochelatase-I. Using RFLP analysis in recombinant inbred lines, the Ferrochelatase-I gene was mapped to chromosome V and that for Ferrochelatase-II to chromosome II. Northern analysis showed that both Ferrochelatase genes are expressed in leaves, stems and flowers, and expression in the leaves is higher in the light than in the dark. However, in roots only Ferrochelatase-I transcripts were detected. High levels of sucrose stimulated expression of Ferrochelatase-I, but had no effect, or repressed slightly, the expression of the Ferrochelatase-II isoform. Import experiments into isolated chloroplasts and mitochondria showed that the Ferrochelatase-II gene encodes a precursor which is imported solely into the chloroplast, in contrast to Ferrochelatase-I which is targeted to both organelles. The significance of these results for haem biosynthesis and the production of haemoproteins, both within the plant cell and in different plant tissues, is discussed.