Determination of nonligand amino acids critical to [4Fe-4S](2+/+) assemblyin ferredoxin maquettes

The prototype ferredoxin maquette, FdM, is a 16-amino acid peptide which ef ficiently incorporates a single [4Fe-4S](2+/+) cluster with spectroscopic a nd electrochemical properties that are typical of natural bacterial ferredo xins. Using this synthetic protein scaffold, we have investigated the role of the nonliganding amino acids in the assembly of the iron-sulfur cluster. In a stepwise fashion, we truncated FdM to a seven-amino acid peptide, FdM -7, which incorporates a cluster spectroscopically identical to FdM but in lower yield, 29% relative to FdM. FdM-7 consists solely of the . CIACGAC . consensus ferredoxin core motif observed in natural protein sequences. Init ially, all of the nonliganding amino acids were substituted for either glyc ine, FdM-7-PolyGly (. CGGCGGC .), or alanine, FdM-7-PolyAla (. CAACAAC .), on the basis of analysis of natural ferredoxin sequences. Both FdM-7-PolyGl y and FdM-7-PolyAla incorporated little [4Fe-4S](2+/+) cluster, 6 and 7%, r espectively. A systematic study of the incorporation of a single isoleucine into each of the four nonliganding positions indicated that placement eith er in the second or in the sixth core motif positions, . CIGCGGC . or . CGG CGIC ., restored the iron-sulfur cluster binding capacity of the peptides t o the level of FdM-7. Incorporation of an isoleucine into the fifth positio n, . CGGCIGC ., which in natural ferredoxins is predominately occupied by a glycine, resulted in a loss of [4Fe-4S] affinity. The substitution of leuc ine, tryptophan, and arginine into the second core motif position illustrat ed the stabilization of the [4Fe-4S] cluster by bulky hydrophobic amino aci ds. Furthermore, the incorporation of a single isoleucine into the second c ore motif position in a 16-amino acid ferredoxin maquette resulted in a 5-f old increase in the level of [4Fe-4S] cluster binding relative to that of t he glycine variant. The protein design rules derived from this study are fu lly consistent with those derived from natural ferredoxin sequence analysis , suggesting they are applicable to both the de novo design and structure-b ased redesign of natural proteins.