Identification and characterization of an inhibitory metal ion-binding site in ferrochelatase.

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.