Substrate interactions with human ferrochelatase

Medlock, Amy E.; Swartz, L.; Dailey, Tamara A.; Dailey, Harry A.; Lanzilotta, William N.

doi:10.1073/pnas.0606144104

Cited by 89 publications

(117 citation statements)

References 37 publications

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“…Based on these results, it has been suggested that the mode of binding of N-MeMP to B. subtilis ferrochelatase may not reflect the proper substrate-binding mode. 22 However, our observation that N-MeMP bound to wild-type B. subtilis ferrochelatase could be metallated while in the complex with the His183Ala variant it could not, suggests strongly that the porphyrin is bound to the protein in a "productive" state.…”

Section: Discussionmentioning

confidence: 84%

“…In human ferrochelatase, the porphyrin is rotated by about 100° and buried by an additional 4.5 Å deeper into the active site cleft, relative to the position of N-MeMP bound to the B. subtilis enzyme. 22,23 The structures of the human enzyme also show that the porphyrin binding cleft is in a closed conformation as compared to the porphyrin-free protein, whereas in B. subtilis ferrochelatase the entrance to the cleft is more open after porphyrin binding. Based on these results, it has been suggested that the mode of binding of N-MeMP to B. subtilis ferrochelatase may not reflect the proper substrate-binding mode.…”

Section: Discussionmentioning

confidence: 99%

“…21 The recently published X-ray structures of the Glu343Lys variant of human ferrochelatase in complex with ppIX, of the lead-inhibited intermediate of the wild-type enzyme, and of the Phe110Ala variant in complex with product (heme) have shown that the porphyrin is rotated by about 100° and buried by an additional 4.5 Å deeper into the active site cleft as compared to the position of N-MeMP in B. subtilis enzyme. 22,23 These differences in porphyrin binding and the apparent differences in the structures of the porphyrin binding pockets of the catalytic antibody and ferrochelatase clearly suggest that the distortions required for the metallation reaction may be imposed by different environments of the substrate.…”

Section: Introductionmentioning

confidence: 99%

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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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Section: Discussionmentioning

confidence: 84%

Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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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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“…Overall these results demonstrate that this residue plays a role in multiple steps in the ferrochelatase mechanism. The dual role of this residue is also apparent from the E343K variant which binds and co-crystallizes with porphyrin (15), but is inactive (1).…”

Section: Eqnmentioning

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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“…However, the mode of binding of the true substrate, protoporphyrin, to human ferrochelatase is different as the substrate is rotated around by 100°and is accommodated approximately 5 Å deeper within the active site. Moreover, only a slight warp is observed in the plane of the macrocycle indicating that just minor ruffling of the tetrapyrrole is required to enhance metal insertion (19,20). The latter, therefore, represents a more accurate substrate/product binding site than that identified by the former inhibitor complex.…”

mentioning

confidence: 99%

Evolution in a family of chelatases facilitated by the introduction of active site asymmetry and protein oligomerization

Romão

Ladakis

Lobo

et al. 2010

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

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The class II chelatases associated with heme, siroheme, and cobalamin biosynthesis are structurally related enzymes that insert a specific metal ion (Fe 2þ or Co 2þ ) into the center of a modified tetrapyrrole (protoporphyrin or sirohydrochlorin). The structures of two related class II enzymes, CbiX S from Archaeoglobus fulgidus and CbiK from Salmonella enterica, that are responsible for the insertion of cobalt along the cobalamin biosynthesis pathway are presented in complex with their metallated product. A further structure of a CbiK from Desulfovibrio vulgaris Hildenborough reveals how cobalt is bound at the active site. The crystal structures show that the binding of sirohydrochlorin is distinctly different to porphyrin binding in the protoporphyrin ferrochelatases and provide a molecular overview of the mechanism of chelation. The structures also give insights into the evolution of chelatase form and function. Finally, the structure of a periplasmic form of Desulfovibrio vulgaris Hildenborough CbiK reveals a novel tetrameric arrangement of its subunits that are stabilized by the presence of a heme b cofactor. Whereas retaining colbaltochelatase activity, this protein has acquired a central cavity with the potential to chaperone or transport metals across the periplasmic space, thereby evolving a new use for an ancient protein subunit.enzyme mechanism | tetrapyrrole biosynthesis

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Substrate interactions with human ferrochelatase

Cited by 89 publications

References 37 publications

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

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

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

Evolution in a family of chelatases facilitated by the introduction of active site asymmetry and protein oligomerization

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