Examination of the activity of carboxyl-terminal chimeric constructs of human and yeast ferrochelatases.
Insertion of ferrous iron into protoporphyrin IX is catalyzed by ferrochelatase (EC 4.99.1.1). Human and Schizosaccharomyces pombe forms of ferrochelatase contain a [2Fe-2S] cluster with three of the four coordinating cysteine ligands located within the 30 carboxyl-terminal residues. Saccharomyces cerevisiae ferrochelatase contains no cluster, but has comparable activity. Truncation mutants of S. cerevisiae lacking either the last 37 or 16 amino acids have no enzyme activity. Chimeric mutants of human, S. cerevisiae, and Sc. pombe ferrochelatase have been created by switching the terminal 10% of the carboxy end of the enzyme. Site-directed mutagenesis has been used to introduce the fourth cysteinyl ligand into chimeric mutants that are 90% S. cerevisiae. Activity was assessed by rescue of Deltahem H, a ferrochelatase deficient strain of Escherichia coli, and by enzyme assays. UV-visible and EPR spectroscopy were used to investigate the presence or absence of the [2Fe-2S] cluster. Only 2 of the 13 chimeric mutants that were constructed produced active enzymes. HYB, which is predominately human with the last 40 amino acids being that of S. cerevisiae, is an active protein which does not contain a [2Fe-2S] cluster. The other active chimeric mutant, HSp, is predominately human ferrochelatase with the last 38 amino acids being that of Sc. pombe ferrochelatase. This active mutant contains a [2Fe-2S] cluster, as verified by UV-visible and EPR spectroscopic techniques. No other chimeric proteins had detectable enzyme activity or a [2Fe-2S] cluster. The data are discussed in terms of structural requirements for cluster stability and the role that the cluster plays for ferrochelatase.
Medlock
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AE
Department of Biochemistry and Molecular Biology, University of Georgia, Athens 30602-2779, USA.
Dailey
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HA
D000595:Amino Acid Sequence; D001483:Base Sequence; D004247:DNA; D005294:Ferrochelatase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000201:enzymology*; D012568:Schizosaccharomyces / Q000201:enzymology*; D017386:Sequence Homology, Amino Acid
D011993:Recombinant Fusion Proteins; D004247:DNA; D005294:Ferrochelatase