Metal Binding to Saccharomyces cerevisiae Ferrochelatase

Abstract

Ferrochelatase is the terminal enzyme in the heme biosynthetic pathway. It catalyzes the insertion of ferrous iron into protoporphyrin IX to produce protoheme IX. The crystal structures of ferrochelatase from Saccharomyces cerevisiae in free form, in complex with Co(II), a substrate metal ion, and in complex with two inhibitors, Cd(II) and Hg(I), are presented in this work. The enzyme is a homodimer, with clear asymmetry between the monomers with regard to the porphyrin binding cleft and the mode of metal binding. The Co(II) and Cd(II) complexes reveal the metal binding site which consists of the invariant amino acids H235, E314, and S275 and solvent molecules. The shortest distance to the metal reveals that amino acid H235 is the primary metal binding residue. A second site with bound Cd(II) was found close to the surface of the molecule, approximately 14 A from H235, with E97, H317, and E326 participating in metal coordination. It is suggested that this site corresponds to the magnesium binding site in Bacillus subtilis ferrochelatase. The latter site is also located at the surface of the molecule and thought to be involved in initial metal binding and regulation.

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

“…6D); this should result in a longer trajectory between the glutamate residues and thus slower metal transfer, which might account for the increase in its K m Fe 2ϩ value. The mode of metal substrate interaction proposed here is consistent with the recent studies of metal binding in yeast ferrochelatase (13,46). In this model, metal binding requires the conserved His 209 (murine numbering) and occurs on the same side of the active site as that of porphyrin binding, whereas the distal residues, including glutamates in the -helical region, play a regulatory role by promoting metal release from the primary binding site (46).…”

“…Indeed, the crystal structures of human and yeast ferrochelatases demonstrated that the active site entrance is delimited by two, oppositely located loops, which are rich in hydrophobic residues (12,36) and were previously hypothesized to facilitate membrane association (12,36). Further, a few positively charged residues in these loops were suggested to interact with the phosphate groups of the membrane phospholipids (13). One of these loops surrounding the active site entrance corresponds to the murine ferrochelatase sequence Gln 248 -Leu 257 , and consistent with the above hypothesis, we observed an accumulation of positively charged amino acid substitutions at the C-terminal hydropho- activity was determined by a continuous fluorimetric assay using protoporphyrin and Fe 2ϩ as substrates.…”

“…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).…”

“…The x-ray crystal structures of ferrochelatases from Bacillus subtilis (10,11), human (12), and yeast Saccharomyces cerevisiae (13) reveal that they share similar folding patterns and active site structures. Whereas B. subtilis ferrochelatase is a monomer (10,11), human (12) and yeast (13) ferrochelatases are homodimers.…”

“…Whereas B. subtilis ferrochelatase is a monomer (10,11), human (12) and yeast (13) ferrochelatases are homodimers. In all cases, each monomeric unit consists of two domains, each of which is folded into a Rossmann-type fold with a four-stranded, parallel ␤-sheet flanked by ␣-helices on both sides (10 -13).…”