Probing the Active-site Residues in Saccharomyces cerevisiae Ferrochelatase by Directed Mutagenesis

Abstract

Ferrochelatase is a mitochondrial inner membranebound 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 sitedirected 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 K m 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 V m and 4 -10-fold increased K m 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.

Probing the Active Site Loop Motif of Murine Ferrochelatase by Random Mutagenesis

“…Although the iron substrate ligands in ferrochelatase remain to be unequivocally identified, nitrogenous and/or oxygenous ligands appear to coordinate the metal substrate (32,42,43 (13). Thus, an increase in the K m Fe 2ϩ of the triple mutant S249A/K250Q/V251C may account for the loss of the stabilization provided by Ser 249 (Table I).…”

“…3). In addition, mutation of the corresponding residue in yeast ferrochelatase (Trp 282 ) yielded a variant (W282L) exhibiting a 10-fold decrease in K m PPIX and an unaltered K m for the metal substrate (32). Actually, the homologous residue in B. subtilis ferrochelatase (Trp 230 ) was shown to stack against the pyrrole ring, thereby stabilizing the position of the porphyrin ring in the crystal structure (11).…”

“…In the wild-type model, the N-terminal loop residues Gln 248 and Ser 249 were buried deeply inside the active site cavity and in very close proximity (about 4 Å) to His 209 and Glu 289 , which were proposed to be essential for catalysis (31)(32)(33)(34). Lys 250 was solvent-exposed, with the side chain projecting to the protein exterior and in close contact with the Gln 260 residue.…”

Chelatases: distort to select?

“…extended X-ray absorption fine structure and Mössbauer), X-ray crystallography and site-directed mutagenesis have been used to characterize the metal-binding sites of ferrochelatase [5,[29][30][31][32][33][34][35]. Mössbauer and X-ray absorption spectroscopies have indicated that ferrochelatase has an ionic coordination environment for Fe 2+ with six nitrogen-and/or oxygen-containing ligands [29,30].…”

Porphyrin Interactions with Wild-type and Mutant Mouse Ferrochelatase

“…Among these residues are a histidine (H183 in B. subtilis ferrochelatase and H207 in the murine mature enzyme) and a glutamate (E264 in B. subtilis ferrochelatase and E287 in the murine mature enzyme). The active site histidine and glutamate have been advocated to be involved in metal substrate binding(l2, 13) and in catalysis (14), respectively. Biophysical characterization of human and mouse ferrochelatase has shown that these enzymes contain a [2Fe-2S] cluster (15,16).…”