title: Cloning and characterization of chironomidae ferrochelatase: copper activation of the purified ferrochelatase.
abstract: All organisms utilize ferrochelatase (EC 4.99.1.1) to catalyze the insertion of ferrous iron into protoposphyrin IX in the terminal step of the heme biosynthetic pathway. Different metal-binding affinity for the enzyme leads to changes in enzyme activity. In this work, we have cloned and over-expressed the enzyme from chironomidae in E. coli. The enzyme was purified and characterized. The recombinant enzyme showed higher enzymatic activity (four-fold increase) in the presence of copper ions and unaffected by calcium ions. Other divalent metal ions including magnesium, manganese, lead, reduced the enzyme activity by >60%. Over 90% of the enzyme activity was inhibited by Zn2+. The sequence alignment of amino acid residues reveals 83% homology with other ferrochelatases. The results of electron proton resonance (EPR) analysis showed that Fe2+ ion was present in the cluster of the recombinant enzyme complex. The recombinant enzyme also contained the [2Fe-2S] center with two-fold higher enzymatic activity than human ferrochelatase.
authors.affiliation: Department of Biochemistry, The Chinese University of Hong Kong, Shatin, NT, Hong Kong.
authors.forename: Yuet Kin
authors.initials: YK
authors.lastname: Leung
authors.forename: Kwong Fai
authors.initials: KF
authors.lastname: Wong
authors.forename: Hung Kay
authors.initials: HK
authors.lastname: Lee
authors.forename: John W
authors.initials: JW
authors.lastname: Ho
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D002413:Cations, Divalent / Q000494:pharmacology; D002683:Chironomidae / Q000201:enzymology*; D003001:Cloning, Molecular; D003300:Copper / Q000494:pharmacology*; D004789:Enzyme Activation / Q000187:drug effects; D005294:Ferrochelatase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D007506:Iron-Sulfur Proteins; D007700:Kinetics; D008670:Metals / Q000494:pharmacology; D016415:Sequence Alignment
chemical_list: D002413:Cations, Divalent; D007506:Iron-Sulfur Proteins; D008670:Metals; D003300:Copper; D005294:Ferrochelatase