Crouse B R, Sellers V M, Finnegan M G, Dailey H A, Johnson M K
Department of Microbiology, University of Georgia, Athens 30602, USA.
Biochemistry. 1996 Dec 17;35(50):16222-9. doi: 10.1021/bi9620114.
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.
人类亚铁螯合酶羧基末端附近的五个半胱氨酸已分别突变为丝氨酸、组氨酸或天冬氨酸残基，以试图确定与[2Fe-2S]簇结合的蛋白质配体。403、406和411位半胱氨酸的突变（C403D、C403H、C406D、C406H、C406S、C411H和C411S突变体）均导致酶失活，通过全细胞电子顺磁共振研究判断，该酶无法组装[2Fe-2S]簇。相比之下，360和395位半胱氨酸突变为丝氨酸（C360S和C395S突变体）并不影响酶活性，所得酶组装的[2Fe-2S]簇在光谱上与野生型酶无法区分。结果表明，哺乳动物亚铁螯合酶30个残基的C末端延伸区中三个保守的半胱氨酸参与连接[2Fe-2S]簇。本文报道了人类亚铁螯合酶无血红素制剂的共振拉曼和变温磁圆二色性研究，根据[2Fe-2S]簇的一个非半胱氨酰、含氧配体，这些光谱得到了最佳解释。与具有完全半胱氨酰连接的类似簇相比，这种异常配位可能解释了簇的不稳定性，因此可能是该簇在哺乳动物亚铁螯合酶中所提出的调节作用所固有的。
