Unraveling the substrate - Metal binding site of ferrochelatase

An X-ray absorption spectroscopic study

Gloria C. Ferreira, Ricardo Franco, Arianna Mangravita, Graham N. George

Research output: Contribution to journalArticle

44 Citations (Scopus)

Abstract

Ferrochelatase (EC 4.99.1.1), the terminal enzyme of the heme biosynthetic pathway, catalyzes the insertion of ferrous iron into the protoporphyrin IX ring. Ferrochelatases can be arbitrarily divided into two broad categories: those with and those without a [2Fe-2S] center. In this work we have used X-ray absorption spectroscopy to investigate the metal ion binding sites of murine and Saccharomyces cerevisiae (yeast) ferrochelatases, which are representatives of the former and latter categories, respectively. Co2+ and Zn2+ complexes of both enzymes were studied, but the Fe2+ complex was only studied for yeast ferrochelatase because the [2Fe-2S] center of the murine enzyme interferes with the analysis. Co2+ and Zn2+ binding to site-directed mutants of the murine enzyme were also studied, in which the highly conserved and potentially metal-coordinating residues H207 and Y220 were substituted by residues that should not coordinate metal (i.e., H207N, H207A, and Y220F). Our experiments indicate four-coordinate zinc with Zn(N/O)3(S/Cl)1 coordination for the yeast and Zn(N/O)2(S/Cl)2 coordination for the wild-type murine enzyme. In contrast to zinc, a six-coordinate site for Co2+ coordinated with oxygen or nitrogen was present in both the yeast and murine (wild-type and mutated) enzymes, with evidence of two histidine ligands in both. Like Co2+, Fe2+ bound to yeast ferrochelatase was coordinated by approximately six oxygen or nitrogen ligands, again with evidence of two histidine ligands. For the murine enzyme, mutation of both H207 and Y220 significantly changed the spectra, indicating a likely role for these residues in metal ion substrate binding. This is in marked disagreement with the conclusions from X-ray crystallographic studies of the human enzyme, and possible reasons for this are discussed.

Original languageEnglish
Pages (from-to)4809-4818
Number of pages10
JournalBiochemistry
Volume41
Issue number15
DOIs
Publication statusPublished - 16 Apr 2002

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