Abstract
Ferrochelatase catalyzes the insertion of ferrous iron into protoporphyrin IX to form heme. The severe metal ion substrate inhibition observed during in vitro studies of the purified enzyme is almost completely eliminated by mutation of an active site histidine residue (His-287, murine ferrochelatase numbering) to leucine and reduced over 2 orders of magnitude by mutation of a nearby conserved phenylalanine residue (Phe-283) to leucine. Elimination of substrate inhibition had no effect on the apparent V(max) for Ni(2+), but the apparent K(m) was increased 100-fold, indicating that the integrity of the inhibitory binding site is important for the enzyme to turn over substrates rapidly at low micromolar metal ion concentrations. The inhibitory site was observed to have a pK(a) value of 8.0, and this value was reduced to 7.5 by the F283L mutation and to 7.4 in a naturally occurring positional variant observed in most bacterial ferrochelatases, murine ferrochelatase H287C. A H287N variant was also found to be substrate-inhibited, but unlike the H287C variant, pH dependence of substrate inhibition was largely eliminated. The data indicate that the inhibitory metal ion-binding site is composed of multiple residues but primarily defined by His-287 and Phe-283 and is crucial for optimal activity at low metal ion concentrations. It is proposed that this binding site may be important for ferrous iron acquisition and desolvation in vivo.
Highlights
In recent years, there has been some debate in the literature fied enzyme is almost completely eliminated by mutation of an active site histidine residue (His-287, murine ferrochelatase numbering) to leucine and reduced over 2 orders of magnitude regarding the mechanism underlying the strict substrate specificity of ferrochelatase in vivo, where only Fe2ϩ, and to a far lesser extent, Zn2ϩ, are incorporated into protoporphyrin IX by mutation of a nearby conserved phenylalanine residue (4 – 6)
Elimination of substrate inhibition had no effect on the apparent Vmax for Ni2؉, but the apparent Km was increased 100-fold, indicating that the integrity of the inhibitory binding site is important for the enzyme to turn over remarkable metal ion substrate promiscuity of ferrochelatases in vitro, which catalyze insertion of a variety of divalent transition metal ion substrates into protoporphyrin IX with kinetic constants similar or equivalent to those for Fe2ϩ
Ferrochelatase can form an immuno-precipitable complex with mitoflatase mutations that lower overall activity below a certain errin [7], an inner mitochondrial membrane iron transporter threshold of about 35% result in erythropoietic protoporphy- [9, 10], and yet the mechanism whereby a trans-membrane ria, a painfully debilitating disease characterized by abnortransporter might deliver iron to a membrane-embedded acmally high levels of protoporphyrin IX [2]
Summary
Dept. of Molecular Medireport, we identify residues comprising the second binding cine, College of Medicine, MDC 7, University of South Florida, Tampa, FL. Of Molecular Medireport, we identify residues comprising the second binding cine, College of Medicine, MDC 7, University of South Florida, Tampa, FL. Site and report the beneficial role of this site in ferrochelatase usf.edu. Ferrochelatase Mechanism usual location of this binding site within a conformationally dynamic active site -helix may offer important clues as to how iron acquisition for heme biosynthesis is realized in vivo
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