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

Abstract

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

The active site of magnesium chelatase

“…⋆ these residues are adjacent to site 1. ‡ Fitted to the Hill equation (6), where s 0.5 is reported instead of K M , and k cat /s 0.5 instead of k cat /K M . ▽ Fitted to the substrate inhibition equation ( 7), where K i = 37.1 ± 7.7 µM.…”

Molecular Determinants of Efficient Cobalt-Substituted Hemoprotein Production in E. coli

“…The location of the productive metal binding site and the face of the porphyrin into which the metal is inserted have been debated in the literature. , Nevertheless, in vitro incorporation of non-native metals by several ferrochelatases (HemH homologues) is well known. ,− To date, it is unclear how chelatases tune their activity for different metals, and many potential effects may contribute depending on the specific protein and metal in question. Distortion of the porphyrin, − specificity-determining active site residues, , differential product inhibition, and exogenous metal delivery are suggested to contribute.…”

“…25,29−32 To date, it is unclear how chelatases tune their activity for different metals, and many potential effects may contribute depending on the specific protein and metal in question. Distortion of the porphyrin, 33−35 specificity-determining active site residues, 36,37 differential product inhibition, 38 and exogenous metal delivery 30 are suggested to contribute.…”