Ferrochelatase π-helix: Implications from examining the role of the conserved π-helix glutamates in porphyrin metalation and product release

Research output: Contribution to journalArticle

Authors

External Institution(s)

  • University of South Florida

Details

Original languageEnglish (US)
Pages (from-to)37-46
Number of pages10
JournalArchives of Biochemistry and Biophysics
Volume644
StatusPublished - Apr 15 2018
Peer-reviewedYes

Abstract

Protoporphyrin ferrochelatase catalyzes the insertion of Fe 2+ 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 pK a of 7.3 ± 0.1, which was assigned to binding of Fe 2+ by deprotonated Glu-314 and Glu-314-assisted Fe 2+ 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 Fe 2+ from the surface of the enzyme. Transition to the closed conformation, with π-helix winding, brings Glu-314-bound Fe 2+ to the active site for incorporation into protoporphyrin.

    Research areas

  • Enzyme mechanism, Erythropoiesis, Heme, Metalloenzyme, Porphyrin, Protoporphyrin ferrochelatase