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

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At the terminal step of heme biosynthesis, ferrochelatase (FECH) catalyzes the insertion of Fe²⁺ into protoporphyrin to form heme. It is located on the inner membrane of the mitochondria of animals. The enzyme inserts divalent metal ions, including Fe²⁺, Co²⁺, and Zn²⁺, into porphyrins in vitro. We have reported that it can remove Fe²⁺ from heme. To characterize the iron-removal reverse activity of FECH, we examined its properties in porcine liver and muscle mitochondria, and isolated porcine FECH cDNA. The amino acid sequence of porcine FECH showed high homology with bovine (91%), human (85%), mouse (87%), and rat (76%) equivalents. It was expressed in Escherichia coli, and purified, and the kinetic properties of the zinc-chelating and iron-removal activities were examined. Both activities peaked at 45 °C, but different optimal pH values, of 7.5–8.0 for zinc-ion insertion and 5.5–6.0 for the reverse reaction were found. The K_m values for mesoporphyrin IX and Zn²⁺ were 6.6 and 1.1 μM, respectively, and the K_m for heme was 5.7 μM. The k_cat value of the forward reaction was about 11-fold higher than that of the reverse reaction, indicating that the enzyme preferably catalyzes the forward reaction rather than the iron-removal reaction. Reverse activity was stimulated by fatty acids and phospholipids, similarly to the case of the forward reaction, indicating that lipids play a role in regulating both enzyme activities.