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 i nto protoporphyrin IX to form protoheme IX. This membrane-bound enzyme has been cloned from a variety of bacteria, plants, mammals, and yeas t. Interestingly, only in mammals has the enzyme been found to contain a [2Fe-2S] cluster. Since the presence of this feature only in mammal s would have significant evolutionary implications and because there h ave been no nonmammalian animal ferrochelatases cloned, expressed, and characterized, we report here the cloning and characterization of fer rochelatase from chicken (Gallus gallus) and an amphibian (Xenopus lae vis). The cDNAs for both of these ferrochelatases were cloned by compl ementation of an Escherichia coli Delta hemH strain. The expressed and purified enzymes were characterized biochemically and both were found to contain [2Fe-2S] clusters. These clusters have spectral characteri stics 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 rea dily destroyed by NO. Sequence analysis of the 3' UTR of both chicken and amphibian cDNAs show that while both have poly(A) tails neither ha ve a consensus polyadenylation signal. The 5' UTR of Xenopus as isolat ed contained 135 bp and possesses no identifiable stem-loop structure. (C) 1998 Academic Press.