Metal Ion Substrate Inhibition of Ferrochelatase

Abstract

Ferrochelatase catalyzes the insertion of ferrous iron into protoporphyrin IX to form heme. Robust kinetic analyses of the reaction mechanism are complicated by the instability of ferrous iron in aqueous solution, particularly at alkaline pH values. At pH 7.00 the half-life for spontaneous oxidation of ferrous ion is approximately 2 min in the absence of metal complexing additives, which is sufficient for direct comparisons of alternative metal ion substrates with iron. These analyses reveal that purified recombinant ferrochelatase from both murine and yeast sources inserts not only ferrous iron but also divalent cobalt, zinc, nickel, and copper into protoporphyrin IX to form the corresponding metalloporphyrins but with considerable mechanistic variability. Ferrous iron is the preferred metal ion substrate in terms of apparent k cat and is also the only metal ion substrate not subject to severe substrate inhibition. Substrate inhibition occurs in the order Cu 2؉ > Zn 2؉ > Co 2؉ > Ni 2؉ and can be alleviated by the addition of metal complexing agents such as ␤-mercaptoethanol or imidazole to the reaction buffer. These data indicate the presence of two catalytically significant metal ion binding sites that may coordinately regulate a selective processivity for the various potential metal ion substrates.

Porcine Ferrochelatase: The Relationship between Iron-Removal Reaction and the Conversion of Heme to Zn-Protoporphyrin

“…The enzyme naturally utilizes ferrous ions as a substrate in vivo, but additionally inserts divalent metal ions such as zinc and cobaltic ions into porphyrin rings in vitro. 1,6) Thus zinc-chelating activity is essential for the formation of the pigment of dried ham in vitro, but the utilization of ferrous ions to form heme in cells is tightly controlled.…”

“…Since divalent metal ions, including Co 2þ , Zn 2þ , and Cu 2þ , can be inserted into porphyrin rings to form the corresponding metalloporphyrins, they inhibited FECH activity to different degrees via competitive inhibition. 6) Heavy metal ions can bind with SH-groups in the catalytic domain of the enzyme, and then the activity is inhibited. 24,25) The present data indicate that the reverse reaction was strongly inhibited by Cu 2þ and Fe 3þ , but not by Fe 2þ , Sn 2þ , or Co 2þ .…”

“…1) Ferrous ions are the target of the enzyme in vivo, while other divalent metal ions, including zinc, cobalt, and tin, are also utilized to form other metalloporphyrins in vitro. 1,6) Both the cDNA and the gene for FECH have been isolated and sequenced from micro-organisms, plants, and animals, including humans, the cow, the mouse, and the rat.…”

The Structure of the Complex between Yeast Frataxin and Ferrochelatase

“…Mature yeast (S. cerevisiae) ferrochelatase was expressed as described for the murine enzyme in Ferreira (48) and purified according to the procedure in Ferreira et al (49), with slight modifications to the composition of the buffers. The buffers and respective compositions were: lysis buffer (20 mM Tris-HCl, pH 8.0, containing 10% glycerol, 1.5% cholate, and 1.5 M sodium chloride), equilibration buffer (20 mM Tris-HCl, pH 8.0, containing 10% glycerol), wash buffer (20 mM Tris-HCl, pH 8.0, containing 10% glycerol and 1.5 M sodium chloride), and elution buffer (20 mM Tris-HCl, pH 8.0, containing 10% glycerol, 1.5 M sodium chloride, and 1.5% cholate).…”

Silver nanoparticles embedded in zeolite membranes: release of silver ions and mechanism of antibacterial action

“…A possible response mechanism to the increased presence of Ag + is that the bacteria decrease the available pools of intracellular iron by increasing expression of ferrochelatase, which tightly binds Fe 2+ , to combat and reduce oxidative stress. 18,19 The downregulation of genes with a role in iron reduction and iron release from proteins further suggests that the bacteria are attempting to control the intracellular levels of Fe 2+ , 20 thereby reducing the amount of iron available for Fenton reactions. Attempts by bacteria to regulate and restore oxidative balance are also suggested by the upregulation of soxR, which is involved in redox sensing and controlling expression of superoxide dismutase and other antioxidants.…”