Characterization of the Fundamental Protein Ligand Requirements of [4Fe-4S]2+/+ Clusters with Sixteen Amino Acid Maquettes
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- 作者：Stephen E. Mulholland ; Brian R. Gibney ; Francesc Rabanal ; and P. Leslie Dutton
- 刊名：Journal of the American Chemical Society
- 出版年：1998
- 出版时间：October 14, 1998
- 年：1998
- 卷：120
- 期：40
- 页码：10296 - 10302
- 全文大小：189K
- 年卷期：v.120,no.40(October 14, 1998)
- ISSN：1520-5126
文摘
A survey of ferredoxin maquettes derived from natural sequences was utilized to obtain a primarysequence competent for [4Fe-4S]2+/+ incorporation for the study of the minimal ligand requirements for clusterassembly. The resultant 16 amino acid ferredoxin maquette (FdM), NH2-KLCEGG
CIACGACGGW-CONH2,incorporates a single [4Fe-4S]2+/+ cluster as evidenced by a cluster titration assayed by electron paramagneticresonance (EPR) spectroscopy. Assembly of the [4Fe-4S] cluster within FdM was kinetically facile (minutestime scale) and the cluster was stable in solution under strictly anaerobic conditions. The four cysteines ofFdM were systematically replaced with nonligating alanine residues resulting in lower yields (10-56% relativeto a FdM control) as quantitiated in the reduced state by EPR spectroscopy demonstrating the necessity foreach in successful cluster incorporation. A single example of a cysteine to leucine modification resulted in alower yield of cluster incorporation equivalent to the analogous alanine replacement indicating a general absenceof steric hindrance for [4Fe-4S] assembly in these small peptides. Pairwise replacement of cysteines withalanines resulted in dramatic loss of yield and cofactor induced assembly of a pair of peptides as evidencedby EPR spectroscopy and size exclusion chromatography, respectively. Alanine substitution of three of thefour cysteines from the FdM sequence resulted in a virtual loss of [4Fe-4S] incorporation ability. Attemptswere made to provide non-cysteine ligands to the incorporated cluster using aspartic acid and histidine residues;however, the lower yield of [4Fe-4S] assembly in these peptides coupled with both the identical EPR spectralparameters and redox potential indicates a lack of observable aspartate or histidine ligation to the clusters.The measured yields of [4Fe-4S] incorporation provide a convenient tool for probing the basic ligandrequirements for the [4Fe-4S]2+/+ cluster in these 16 amino acid ferredoxin maquettes.
CIACGACGGW-CONH2,incorporates a single [4Fe-4S]2+/+ cluster as evidenced by a cluster titration assayed by electron paramagneticresonance (EPR) spectroscopy. Assembly of the [4Fe-4S] cluster within FdM was kinetically facile (minutestime scale) and the cluster was stable in solution under strictly anaerobic conditions. The four cysteines ofFdM were systematically replaced with nonligating alanine residues resulting in lower yields (10-56% relativeto a FdM control) as quantitiated in the reduced state by EPR spectroscopy demonstrating the necessity foreach in successful cluster incorporation. A single example of a cysteine to leucine modification resulted in alower yield of cluster incorporation equivalent to the analogous alanine replacement indicating a general absenceof steric hindrance for [4Fe-4S] assembly in these small peptides. Pairwise replacement of cysteines withalanines resulted in dramatic loss of yield and cofactor induced assembly of a pair of peptides as evidencedby EPR spectroscopy and size exclusion chromatography, respectively. Alanine substitution of three of thefour cysteines from the FdM sequence resulted in a virtual loss of [4Fe-4S] incorporation ability. Attemptswere made to provide non-cysteine ligands to the incorporated cluster using aspartic acid and histidine residues;however, the lower yield of [4Fe-4S] assembly in these peptides coupled with both the identical EPR spectralparameters and redox potential indicates a lack of observable aspartate or histidine ligation to the clusters.The measured yields of [4Fe-4S] incorporation provide a convenient tool for probing the basic ligandrequirements for the [4Fe-4S]2+/+ cluster in these 16 amino acid ferredoxin maquettes.