Revisiting catalytic His and Glu residues in coproporphyrin ferrochelatase - unexpected activities of active site variants

The identification of the coproporphyrin-dependent heme biosynthetic pathway, which is used almost exclusively by monoderm bacteria in 2015 by Dailey et al. triggered studies aimed at investigating the enzymes involved in this pathway that were originally assigned to the protoporphyrin-dependent hem...

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Bibliographic Details
Published in:The FEBS journal 2024-05, Vol.291 (10), p.2260-2272
Main Authors: Gabler, Thomas, Dali, Andrea, Bellei, Marzia, Sebastiani, Federico, Becucci, Maurizio, Battistuzzi, Gianantonio, Furtmüller, Paul Georg, Smulevich, Giulietta, Hofbauer, Stefan
Format: Article
Language:English
Subjects:
Bacterial Proteins - chemistry
Bacterial Proteins - genetics
Bacterial Proteins - metabolism
Catalysis
Catalytic Domain
Coproporphyrins - chemistry
Coproporphyrins - metabolism
Ferrochelatase
Ferrochelatase - chemistry
Ferrochelatase - genetics
Ferrochelatase - metabolism
Glutamic Acid - chemistry
Glutamic Acid - genetics
Glutamic Acid - metabolism
Heme
Heme - chemistry
Heme - metabolism
Histidine - chemistry
Histidine - genetics
Histidine - metabolism
Kinetics
Listeria monocytogenes
Models, Molecular
Oxidation
Oxidation-Reduction
Physiology
Protoporphyrin
Protoporphyrin IX
Protoporphyrins - chemistry
Protoporphyrins - metabolism
Reaction mechanisms
Residues
Structure-function relationships
Substrate Specificity
Substrates
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Summary:The identification of the coproporphyrin-dependent heme biosynthetic pathway, which is used almost exclusively by monoderm bacteria in 2015 by Dailey et al. triggered studies aimed at investigating the enzymes involved in this pathway that were originally assigned to the protoporphyrin-dependent heme biosynthetic pathway. Here, we revisit the active site of coproporphyrin ferrochelatase by a biophysical and biochemical investigation using the physiological substrate coproporphyrin III, which in contrast to the previously used substrate protoporphyrin IX has four propionate substituents and no vinyl groups. In particular, we have compared the reactivity of wild-type coproporphyrin ferrochelatase from the firmicute Listeria monocytogenes with those of variants, namely, His182Ala (H182A) and Glu263Gln (E263Q), involving two key active site residues. Interestingly, both variants are active only toward the physiological substrate coproporphyrin III but inactive toward protoporphyrin IX. In addition, E263 exchange impairs the final oxidation step from ferrous coproheme to ferric coproheme. The characteristics of the active site in the context of the residues involved and the substrate binding properties are discussed here using structural and functional means, providing a further contribution to the deciphering of this enigmatic reaction mechanism.
ISSN:1742-464X
1742-4658
1742-4658
DOI:10.1111/febs.17101