title: Unraveling the substrate-metal binding site of ferrochelatase: an X-ray absorption spectroscopic study.
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. Co(2+) and Zn(2+) complexes of both enzymes were studied, but the Fe(2+) complex was only studied for yeast ferrochelatase because the [2Fe-2S] center of the murine enzyme interferes with the analysis. Co(2+) and Zn(2+) 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 Co(2+) 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 Co(2+), Fe(2+) 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.
authors.affiliation: Department of Biochemistry and Molecular Biology, College of Medicine, Institute for Biomolecular Science and H. Lee Moffitt Cancer Center and Research Institute, University of South Florida, Tampa, Florida 33612, USA. gferreir@hsc.usf.edu
authors.forename: Gloria C
authors.initials: GC
authors.lastname: Ferreira
authors.forename: Ricardo
authors.initials: R
authors.lastname: Franco
authors.forename: Arianna
authors.initials: A
authors.lastname: Mangravita
authors.forename: Graham N
authors.initials: GN
authors.lastname: George
mesh_terms: D019943:Amino Acid Substitution; D000818:Animals; D016477:Artifacts; D001665:Binding Sites; D002413:Cations, Divalent / Q000378:metabolism; D003035:Cobalt / Q000378:metabolism; D017124:Conserved Sequence; D003300:Copper / Q000378:metabolism; D005294:Ferrochelatase / Q000737:chemistry* / Q000378:metabolism*; D007501:Iron / Q000378:metabolism; D007506:Iron-Sulfur Proteins / Q000737:chemistry; D007700:Kinetics; D008670:Metals / Q000378:metabolism*; D051379:Mice; D008958:Models, Molecular; D010957:Plasmids; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000201:enzymology; D013052:Spectrometry, X-Ray Emission / Q000379:methods; D013057:Spectrum Analysis / Q000379:methods; D015032:Zinc / Q000378:metabolism
chemical_list: D002413:Cations, Divalent; D007506:Iron-Sulfur Proteins; D008670:Metals; D011994:Recombinant Proteins; D003035:Cobalt; D003300:Copper; D007501:Iron; D005294:Ferrochelatase; D015032:Zinc