Y13C AZOTOBACTER-VINELANDII FERREDOXIN-I - A DESIGNED [FE-S] LIGAND MOTIF CONTAINS A CYSTEINE PERSULFIDE

Ferredoxins that contain [4Fe-4S](2+/+) clusters often obtain three of their four cysteine ligands from a highly conserved CysXXCysXXCys seq uence motif. Little is known about the in vitro assembly of these clus ters and the role that this sequence motif plays in that process, In t his study, we have used structure as a guide in attempts to direct the formation of a [4Fe-4S](2+/+) in the [3Fe-4S](+/0) location of native (7Fe) Azotobacter vinelandii ferredoxin I (AvFdI) by providing the co rrect three-dimensional orientation of cysteine ligands without introd ucing a CysXXCysXXCys motif. Tyr(13) of AvFdr occupies the position of the fourth ligating cysteine in the homologous and structurally chara cterized 8Fe ferredoxin from Peptococcus aerogenes and a Y13C variant of AvFdI could be easily modeled as an 8Fe protein, However, character ization of purified Y13C FdI by UV-visible spectra, circular dichroism , electron paramagnetic resonance spectroscopies, and by x-fay crystal lography revealed that the protein failed to use the introduced cystei ne as a ligand and retained its [3Fe-4S](+/0) cluster. Further, electr ochemical characterization showed that the redox potential and pH beha vior of the cluster were unaffected by the substitution of Tyr by Cys, Although Y13C FdI is functional in vivo it does differ significantly from native FdI in that it is extremely unstable in the reduced state possibly due to increased solvent exposure of the [3Fe-4S](0) cluster. Surprisingly, the rr-ray structure showed that the introduced cystein e was modified to become a persulfide, This modification may have occu rred in vivo via the action of NifS, which is known to be expressed un der the growth conditions used. It is interesting to note that neither of the two free cysteines present in FdI was modified, Thus, if NiFS is involved in modifying the introduced cysteine there must be specifi city to the reaction.