Day A L, Parsons B M, Dailey H A
Department of Biochemistry and Molecular Biology, University of Georgia, Athens, Georgia, 30602-7229, USA.
Arch Biochem Biophys. 1998 Nov 15;359(2):160-9. doi: 10.1006/abbi.1998.0910.
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.
亚铁螯合酶（EC 4.99.1.1）催化亚铁插入原卟啉IX中以形成原血红素IX。这种膜结合酶已从多种细菌、植物、哺乳动物和酵母中克隆出来。有趣的是，仅在哺乳动物中发现该酶含有一个[2Fe-2S]簇。由于这一特征仅在哺乳动物中存在具有重要的进化意义，并且由于尚未克隆、表达和表征非哺乳动物动物的亚铁螯合酶，我们在此报告鸡（原鸡）和一种两栖动物（非洲爪蟾）亚铁螯合酶的克隆和表征。这两种亚铁螯合酶的cDNA通过互补大肠杆菌ΔhemH菌株进行克隆。对表达和纯化的酶进行了生化表征，发现两者均含有[2Fe-2S]簇。这些簇的光谱特征与人类亚铁螯合酶的基本相同，尽管它们的电子顺磁共振光谱与人类的明显不同。鸡和两栖动物亚铁螯合酶的[2Fe-2S]簇很容易被一氧化氮破坏。对鸡和两栖动物cDNA的3'非翻译区的序列分析表明，虽然两者都有多聚腺苷酸尾，但都没有共有多聚腺苷酸化信号。分离得到非洲爪蟾的5'非翻译区含有135个碱基对，且不具有可识别的茎环结构。
