CHARACTERIZATION OF THE FUNDAMENTAL PROTEIN LIGAND REQUIREMENTS OF [4FE-4S](2+ +) CLUSTERS WITH 16 AMINO-ACID MAQUETTES/

A survey of ferredoxin maquettes derived from natural sequences was ut ilized to obtain a primary sequence competent for [4Fe-4S](2+/+) incor poration for the study of the minimal ligand requirements for cluster assembly. The resultant 16 amino acid ferredoxin maquette (FdM), NH2-K LCEGG . CIACGAC . GGW-CONH2, incorporates a single [4Fe-4S](2+/+) clus ter as evidenced by a cluster titration assayed by electron paramagnet ic resonance (EPR) spectroscopy. Assembly of the [4Fe-4S] cluster with in FdM was kinetically facile (minutes time scale) and the cluster was stable in solution under strictly anaerobic conditions. The four cyst eines of FdM were systematically replaced with nonligating alanine res idues resulting in lower yields (10-56% relative to a FdM control) as quantitiated in the reduced state by EPR spectroscopy demonstrating th e necessity for each in successful cluster incorporation. A single exa mple of a cysteine to leucine modification resulted in a lower yield o f cluster incorporation equivalent to the analogous alanine replacemen t indicating a general absence of steric hindrance for [4Fe-4S] assemb ly in these small peptides. Pairwise replacement of cysteines with ala nines resulted in dramatic loss of yield and cofactor induced assembly of a pair of peptides as evidenced by EPR spectroscopy and size exclu sion chromatography, respectively. Alanine substitution of three of th e four cysteines from the FdM sequence resulted in a virtual loss of [ 4Fe-4S] incorporation ability. Attempts were made to provide non-cyste ine ligands to the incorporated cluster using aspartic acid and histid ine residues; however, the lower yield of [4Fe-4S] assembly in these p eptides coupled with both the identical EPR spectral parameters and re dox potential indicates a lack of observable aspartate or histidine li gation to the clusters. The measured yields of [4Fe-4S] incorporation provide a convenient tool for probing the basic ligand requirements fo r the [4Fe-4S](2+/+) cluster in these 16 amino acid ferredoxin maquett es.