title: Probing the active-site residues in Saccharomyces cerevisiae ferrochelatase by directed mutagenesis. In vivo and in vitro analyses.
abstract: Ferrochelatase is a mitochondrial inner membrane-bound enzyme that catalyzes the insertion of ferrous iron into protoporphyrin, the terminal step in protoheme biosynthesis. The functional/structural roles of 10 invariant amino acid residues were investigated by site-directed mutagenesis in the yeast Saccharomyces cerevisiae ferrochelatase. The mutant enzymes were expressed in a yeast strain lacking the ferrochelatase gene HEM15 and in Escherichia coli. The kinetic parameters of the mutant enzymes were determined for the enzymes associated with the yeast membranes and the enzymes in the bacterial soluble fraction. They were compared with the in vivo functioning of the mutant enzymes. The main conclusions are the following. Glu-314 is critical for catalysis, and we suggest that it is the base responsible for abstracting the N-pyrrole proton(s). His-235 is essential for metal binding. Asp-246 and Tyr-248 are also involved in metal binding in a synergistic manner. The Km for protoporphyrin was also increased in the H235L, D246A, and Y248L mutants, suggesting that the binding sites of the two substrates are not independent of each other. The R87A, Y95L, Q111E, Q273E, W282L, and F308A mutants had 1.2-2-fold increased Vm and 4-10-fold increased Km values for protoporphyrin, but the amount of heme made in vivo was 10-100% of the normal value. These mutations probably affected the geometry of the active center, resulting in improper positioning of protoporphyrin.
authors.affiliation: Institute of Biochemistry and Biophysics, Polish Academy of Sciences, Warsaw, Poland.
authors.forename: M
authors.initials: M
authors.lastname: Gora
authors.forename: E
authors.initials: E
authors.lastname: Grzybowska
authors.forename: J
authors.initials: J
authors.lastname: Rytka
authors.forename: R
authors.initials: R
authors.lastname: Labbe-Bois
mesh_terms: D000595:Amino Acid Sequence; D001483:Base Sequence; D001665:Binding Sites; D005294:Ferrochelatase / Q000737:chemistry* / Q000378:metabolism; D007700:Kinetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D012441:Saccharomyces cerevisiae / Q000201:enzymology*
chemical_list: D005294:Ferrochelatase