| Abstract
| - 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 ofthe four coordinating cysteine ligands located within the 30 carboxyl-terminal residues. Saccharomycescerevisiae 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 ofhuman, S. cerevisiae, and Sc. pombe ferrochelatase have been created by switching the terminal 10% ofthe carboxy end of the enzyme. Site-directed mutagenesis has been used to introduce the fourth cysteinylligand into chimeric mutants that are 90% S. cerevisiae. Activity was assessed by rescue of Δhem H, aferrochelatase deficient strain of Escherichia coli, and by enzyme assays. UV−visible and EPR spectroscopywere used to investigate the presence or absence of the [2Fe-2S] cluster. Only 2 of the 13 chimeric mutantsthat were constructed produced active enzymes. HYB, which is predominately human with the last 40amino 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 aminoacids being that of Sc. pombe ferrochelatase. This active mutant contains a [2Fe-2S] cluster, as verifiedby UV−visible and EPR spectroscopic techniques. No other chimeric proteins had detectable enzymeactivity or a [2Fe-2S] cluster. The data are discussed in terms of structural requirements for cluster stabilityand the role that the cluster plays for ferrochelatase.
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