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

• Published in: Archives of biochemistry and biophysics Vol. 644; pp. 37 - 46
• Main Authors: Gillam, Mallory E., Hunter, Gregory A., Ferreira, Gloria C.
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 15.04.2018
• Subjects: Enzyme mechanism; Erythropoiesis; Heme; Metalloenzyme; Porphyrin; Protoporphyrin ferrochelatase; Erythropoiesis; MOPS; PIPBS; Enzyme mechanism; Porphyrin; Protoporphyrin ferrochelatase; Heme; PPIX; Metalloenzyme
• ISSN: 0003-9861; 1096-0384
• DOI: 10.1016/j.abb.2018.02.015

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 ± 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. [Display omitted]