Ferrochelatase π-helix: Implications from examining the role of the conserved π-helix glutamates in porphyrin metalation and product release.
Arch Biochem Biophys
; 644: 37-46, 2018 04 15.
Article
em En
| MEDLINE
| ID: mdl-29481781
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⯱â¯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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Base de dados:
MEDLINE
Assunto principal:
Protoporfirinas
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Saccharomyces cerevisiae
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Bacillus subtilis
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Proteínas de Bactérias
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Proteínas de Saccharomyces cerevisiae
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Ferroquelatase
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Ferro
Limite:
Animals
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Humans
Idioma:
En
Ano de publicação:
2018
Tipo de documento:
Article