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Site-directed mutagenesis and spectroscopic characterization of human ferrochelatase: identification of residues coordinating the [2Fe-2S] cluster

, : Site-directed mutagenesis and spectroscopic characterization of human ferrochelatase: identification of residues coordinating the [2Fe-2S] cluster. Biochemistry 35(50): 16222-9

The five cysteines closest to the carboxyl terminus of human ferrochelatase have been individually mutated to serine, histidine, or aspartate residues in an attempt to identify the protein ligands to the [2Fe-2S] cluster. Mutations of cysteines at positions 403, 406, and 411 (C403D, C403H, C406D, C406H, C406S, C411H, and C411S mutants) all resulted in inactive enzyme that failed to assemble the [2Fe-2S] cluster as judged by whole-cell EPR studies. In contrast, mutation of the cysteines at positions 360 and 395 to serines (C360S and C395S mutants) did not affect the enzymatic activity, and the resulting enzyme assembled a [2Fe-2S] cluster that was spectroscopically indistinguishable from the wild-type enzyme. The results indicate that three of the conserved cysteines in the 30-residue C-terminal extension of mammalian ferrochelatase are involved in ligating the [2Fe-2S] cluster. Resonance Raman and variable-temperature magnetic circular dichroism studies of heme-free preparations of human ferrochelatase are reported, and the spectra are best interpreted in terms of one non-cysteinyl, oxygenic ligand for the [2Fe-2S] cluster. Such anomalous coordination could account for the cluster lability compared to similar clusters with complete cysteinyl ligation and hence may be intrinsic to the proposed regulatory role for this cluster in mammalian ferrochelatases.


PMID: 8973195

DOI: 10.1021/bi9620114

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