Cloning and characterization of Gallus and Xenopus ferrochelatases: presence of the [2Fe-2S] cluster in nonmammalian ferrochelatase.
Ferrochelatase (EC 4.99.1.1) catalyzes the insertion of ferrous iron into protoporphyrin IX to form protoheme IX. This membrane-bound enzyme has been cloned from a variety of bacteria, plants, mammals, and yeast. Interestingly, only in mammals has the enzyme been found to contain a [2Fe-2S] cluster. Since the presence of this feature only in mammals would have significant evolutionary implications and because there have been no nonmammalian animal ferrochelatases cloned, expressed, and characterized, we report here the cloning and characterization of ferrochelatase from chicken (Gallus gallus) and an amphibian (Xenopus laevis). The cDNAs for both of these ferrochelatases were cloned by complementation of an Escherichia coli DeltahemH strain. The expressed and purified enzymes were characterized biochemically and both were found to contain [2Fe-2S] clusters. These clusters have spectral characteristics essentially identical to those of human ferrochelatase, although their EPR spectra are recognizably distinct from the human one. The [2Fe-2S] clusters of both chicken and amphibian ferrochelatases are readily destroyed by NO. Sequence analysis of the 3' UTR of both chicken and amphibian cDNAs show that while both have poly(A) tails neither have a consensus polyadenylation signal. The 5' UTR of Xenopus as isolated contained 135 bp and possesses no identifiable stem-loop structure.
Day
A L
AL
Department of Biochemistry and Molecular Biology, University of Georgia, Athens, Georgia, 30602-7229, USA.
Parsons
B M
BM
Dailey
H A
HA
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites / Q000235:genetics; D002417:Cattle; D002645:Chickens; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000032:analysis; D004926:Escherichia coli / Q000235:genetics; D005294:Ferrochelatase / Q000737:chemistry* / Q000235:genetics*; D005816:Genetic Complementation Test; D005822:Genetic Vectors; D006801:Humans; D007501:Iron / Q000378:metabolism*; D051379:Mice; D008969:Molecular Sequence Data; D017422:Sequence Analysis, DNA; D017386:Sequence Homology, Amino Acid; D013455:Sulfur / Q000378:metabolism*; D014982:Xenopus laevis
D018076:DNA, Complementary; D013455:Sulfur; D007501:Iron; D005294:Ferrochelatase