Modulation of inhibition of ferrochelatase by <i>N</i>-methylprotoporphyrin

Shi, Zhongyuan; Ferreira, Glória C.

doi:10.1042/bj20060753

Cited by 17 publications

(17 citation statements)

References 30 publications

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“…This behavior requires that significant amounts of the EZnD IX complex exist in the steady state and therefore that ZnD IX disassociation is partially rate-limiting. This result is thus in agreement with previous transient kinetic studies using iron (16) and zinc (33,34) as metal ion substrates. This conclusion can be contrasted to previous work with the bovine enzyme (35) where steady-state inhibition studies using both product and alternative substrate demonstrated an ordered mechanism where iron bound first.…”

Section: Discussionsupporting

confidence: 83%

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: Discussionsupporting

confidence: 83%

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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“…A pre-steady state burst has recently been observed in the murine ferrochelatase catalyzed insertion of zinc into protoporphyrin IX (27); the transient was used to make a detailed study of binding of the inhibitor N-methyl protoporphyrin. However, rate constants for metal ion insertion in the active site were not determined.…”

Section: Discussionmentioning

confidence: 99%

“…1, 2,3), but there are very few reports of transient kinetics (3,27). Most functional analysis and assessment of the significance of mutated residues has relied on steady-state information.…”

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confidence: 99%

Direct Measurement of Metal Ion Chelation in the Active Site of Human Ferrochelatase

et al. 2007

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The final step in heme biosynthesis, insertion of ferrous iron into protoporphyrin IX, is catalyzed by protoporphyrin IX ferrochelatase (E.C. 4.99.1.1). It is demonstrated that the pre-steady state human ferrochelatase (R115L) shows a stoichiometric burst of product formation and substrate consumption, consistent with a rate determining step following metal-ion chelation. Detailed analysis shows that chelation requires at least two steps, rapid binding followed by a slower (k ca. 1 s −1 ) irreversible step, provisionally assigned to metal ion chelation. Comparison with steady-state data reveals that the rate-determining step in the overall reaction, converting free porphyrin to free metalloporphyrin, occurs after chelation and is most probably product release. We have measured rate constants for significant steps on the enzyme and demonstrate that metal-ion chelation, with a rate constant of 0.96 s −1 , is around 10 times faster than the rate determining step in the steady-state (k cat 0.1 s −1 ). The effect of an additional E343D mutation is apparent at multiple stages in the reaction cycle with a seven fold drop in k cat and three fold drop in k chel . This conservative mutation primarily affects events occurring after metal ion chelation. Further evaluation of structure-function data on site-directed mutants will therefore require both steady state and pre-steady state approaches. Keywords porphyrin biosynthesis; ferrochelatase; transient kineticsThe final step in heme biosynthesis, insertion of ferrous iron into protoporphyrin IX, is catalyzed by protoporphyrin IX ferrochelatase (protoheme ferro-lyase, E.C. 4.99.1.1, the human enzyme is hereafter referred to as ferrochelatase). The mechanisms of biological iron chelation have proved to be of great interest with a range of studies examining reaction kinetics (1-3), spectroscopy of bound intermediates (4-6), sensitivity of the reaction to structural variation (1,3), and behavior of the enzyme analogues, including antibodies (4,7-9) and both DNA and RNA (10-12) that also catalyze metal ion insertion into porphyrins. Additionally crystal structures of free enzyme (13,14), as well as with bound metal substrate , bound porphyrin substrate (15) and a tight-binding competitive inhibitor (16) should support confident interpretation of the link between structure and function in this system. In fact, controversy remains over the role of individual residues in the reaction mechanism; compare for example the suggested metal ion binding site of Sellers et al. (1) with that proposed by Gora et al. † Funded by the BBSRC (UK).

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“…26 The emission at 347 nm from 0.20 μM ferrochelatase excited at 288 nm was monitored as the concentration of dSDP was increased, using a Fluoro-Max-2 fluorimeter (Jobon Yvon Spex). Measurements were carried out on triplicate samples for each condition and enzyme.…”

Section: Fluorescence Titration Measurementsmentioning

confidence: 99%

“…While dSDP is a weak inhibitor of bovine and murine ferrochelatases with K i and k d in the micromolar range, Nmethylprotoporphyrin IX (N-MePP) has K i and k d in the nanomolar range. [24][25][26] The structure of the complex with dSDP reveals for the first time the mechanism of inhibition of ferrochelatase by inhibitors with bulky substituents at positions 2 and 4 of the porphyrin ring. His183 is one of the few invariant residues in the ferrochelatase family.…”

Section: Introductionmentioning

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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Modulation of inhibition of ferrochelatase by N-methylprotoporphyrin

Cited by 17 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

Direct Measurement of Metal Ion Chelation in the Active Site of Human Ferrochelatase

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

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