Porcine ferrochelatase: the relationship between iron-removal reaction and the conversion of heme to Zn-protoporphyrin.
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 degrees 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 microM, respectively, and the K(m) for heme was 5.7 microM. 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.
Chau
Tuan Thanh
TT
Department of Biotechnology, Kyoto Institute of Technology.
Ishigaki
Mutsumi
M
Kataoka
Takao
T
Taketani
Shigeru
S
D000595:Amino Acid Sequence; D000818:Animals; D055162:Biocatalysis; D002417:Cattle; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000235:genetics; D005294:Ferrochelatase / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D006418:Heme / Q000378:metabolism*; D006801:Humans; D007501:Iron / Q000378:metabolism*; D007700:Kinetics; D008099:Liver / Q000201:enzymology; D051379:Mice; D008928:Mitochondria / Q000201:enzymology; D008969:Molecular Sequence Data; D009132:Muscles / Q000166:cytology; D011524:Protoporphyrins / Q000378:metabolism*; D051381:Rats; D013552:Swine
D018076:DNA, Complementary; D011524:Protoporphyrins; C017803:zinc protoporphyrin; D006418:Heme; D007501:Iron; D005294:Ferrochelatase