Crosstalk between metal ions in Bacillus subtilis ferrochelatase

Hansson, Mattias; Lindstam, Mats; Hansson, Mats

doi:10.1007/s00775-006-0080-2

Cited by 14 publications

(14 citation statements)

References 30 publications

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“…However, reasonable objections can be made to the use of zinc; it is not the physiological substrate, and conclusions are hard to translate between metal ion substrates (1). This concern is strongly supported by the recent demonstrations with a variety of metal ions showing that alternative metal ion binding sites can inhibit or activate the reaction (3,23). In addition, the relatively fast uncatalyzed insertion of zinc into porphyrins can complicate the observed kinetics.…”

Section: Discussionmentioning

confidence: 66%

Metal Ion Selectivity and Substrate Inhibition in the Metal Ion Chelation Catalyzed by Human Ferrochelatase

Davidson

Chesters

Reid

2009

Journal of Biological Chemistry

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Protoporphyrin IX ferrochelatase (EC 4.99.1.1) catalyzes the terminal step in the heme biosynthetic pathway, the insertion of ferrous iron into protoporphyrin IX. Ferrochelatase shows specificity, in vitro, for multiple metal ion substrates and exhibits substrate inhibition in the case of zinc, copper, cobalt, and nickel. Zinc is the most biologically significant of these; when iron is depleted, zinc porphyrins are formed physiologically. Examining the k cat /K m app ratios for zinc and iron reveals that, in vitro, zinc is the preferred substrate at all concentrations of porphyrin. This is not the observed biological specificity, where zinc porphyrins are abnormal; these data argue for the existence of a specific iron delivery mechanism in vivo. We demonstrate that zinc acts as an uncompetitive substrate inhibitor, suggesting that ferrochelatase acts via an ordered pathway. Steady-state characterization demonstrates that the apparent k cat depends on zinc and shows substrate inhibition. Although porphyrin substrate is not inhibitory, zinc inhibition is enhanced by increasing porphyrin concentration. This indicates that zinc inhibits by binding to an enzyme-product complex (EZnD IX ) and is likely to be the second substrate in an ordered mechanism. Our analysis shows that substrate inhibition by zinc is not a mechanism that can promote specificity for iron over zinc, but is instead one that will reduce the production of all metalloporphyrins in the presence of high concentrations of zinc.Ferrochelatase (EC 4.99.1.1), the final enzyme of heme biosynthesis, catalyzes the insertion of ferrous iron into protoporphyrin IX (1); this reaction is commonly proposed to involve a distorted porphyrin intermediate (1,2). In vitro, ferrochelatase has a broad metal ion specificity with insertion of Zn 2ϩ , Co 2ϩ , and Cu 2ϩ having also been observed (1, 3). Metal ion specificity is species-dependent; the ferrochelatase from Bacillus subtilis accepts Cu 2ϩ but not Co 2ϩ as a substrate (4), in contrast to the widely reported specificity of most other ferrochelatases. Interestingly, it has recently been demonstrated that the poor activity toward Cu 2ϩ as a substrate, in the case of the murine and yeast ferrochelatases at least, arises from substantial substrate inhibition (3). Of the competing metal ions, Zn 2ϩ is perhaps the most biologically significant; under conditions of iron depletion or lead poisoning, zinc porphyrins can be formed physiologically (5), and the presence of zinc porphyrins in human blood can be used as a diagnostic test for lead poisoning (6). It has been suggested that the metal ion specificity of ferrochelatase is determined by the extent of porphyrin distortion in the active site of the enzyme (2). More recent crystallography has shown that some metal ion inhibitors are inserted into porphyrins, and the inhibition therefore arises from reduced product release (7).Crystal structures of free enzyme (8, 9), as well as enzyme with metal (10, 11), inhibitors (12, 13), porphyrin substrate (14), and a range of po...

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Section: Discussionmentioning

confidence: 66%

Metal Ion Selectivity and Substrate Inhibition in the Metal Ion Chelation Catalyzed by Human Ferrochelatase

Davidson

Chesters

Reid

2009

Journal of Biological Chemistry

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“…Many of the enzymes involved in protection from oxidative stress contain haem groups and consequently the hemAXCDBL‐2 operon (BA4693‐98), encoding enzymes required in the early part of the haem biosynthetic pathway from glutamate to tetrapyrroleuropor‐phyrinogenIII (UroIll), are upregulated 56. The final step in the pathway, involving the incorporation of Fe[II], is catalysed by ferrochelatase 57. B. anthracis has two genes encoding ferrochelatases, hemH‐1 and hemH‐2 , but only the latter, transcriptionally linked to katB (see above), is induced in response to peroxide stress.…”

Section: Resultsmentioning

confidence: 99%

Combined proteomic and transcriptomic analysis of the response of Bacillus anthracis to oxidative stress

Pohl

Aldridge

et al. 2011

Proteomics

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The endospore-forming Gram-positive pathogen Bacillus anthracis is responsible for the usually fatal disease, inhalational anthrax. The success of this pathogen is dependent on its ability to subvert elements of the innate immune system of its animal hosts. B. anthracis spores, which are the main infective agent, are engulfed and germinate in patrolling alveolar macrophages. In order for the infection to progress, the resulting vegetative cells must resist the antimicrobial oxidative burst mounted by the host NADPH oxidase complex. The response of B. anthracis to this and other macrophage-related stresses is therefore of major importance to the success of this pathogen, and consequently we have analysed the superoxide and peroxide stress stimulons of B. anthracis strain UM23C1-2 by means of a combined transcriptomics and proteomics approach. The results show distinct patterns of expression in response to paraquat (endogenous superoxide) and hydrogen peroxide stress. While the main response to paraquat is the induction of iron uptake pathways, the response to peroxide predominantly involves the induction of protection and repair mechanisms. Comparisons between the responses of B. anthracis and related soil bacterium, B. subtilis, reveal differences that are likely to be relevant to their respective habitats.

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“…43 Site-directed mutagenesis was previously performed to construct the plasmid encoding the His183Ala variant of B. subtilis ferrochelatase. 32 Crystals of the His183Ala ferrochelatase complexed with N-MeMP (Frontier Scientific, Logan, UT, USA) were initially grown by vapor diffusion, 20 and subsequently optimized by seeding techniques.…”

Section: Methodsmentioning

confidence: 99%

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

Karlberg

Hansson

Yengo

et al. 2008

Journal of Molecular Biology

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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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Crosstalk between metal ions in Bacillus subtilis ferrochelatase

Cited by 14 publications

References 30 publications

Metal Ion Selectivity and Substrate Inhibition in the Metal Ion Chelation Catalyzed by Human Ferrochelatase

Metal Ion Selectivity and Substrate Inhibition in the Metal Ion Chelation Catalyzed by Human Ferrochelatase

Combined proteomic and transcriptomic analysis of the response of Bacillus anthracis to oxidative stress

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

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