1ftc
From Proteopedia
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'''Y13C MUTANT OF AZOTOBACTER VINELANDII FDI''' | '''Y13C MUTANT OF AZOTOBACTER VINELANDII FDI''' | ||
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[[Category: Prasad, G S.]] | [[Category: Prasad, G S.]] | ||
[[Category: Stout, C D.]] | [[Category: Stout, C D.]] | ||
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| - | + | ''Page seeded by [http://oca.weizmann.ac.il/oca OCA ] on Fri May 2 16:44:35 2008'' | |
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Revision as of 13:44, 2 May 2008
Y13C MUTANT OF AZOTOBACTER VINELANDII FDI
Overview
Ferredoxins that contain [4Fe-4S]2+/+ clusters often obtain three of their four cysteine ligands from a highly conserved CysXXCysXXCys sequence motif. Little is known about the in vivo assembly of these clusters and the role that this sequence motif plays in that process. In this 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 correct three-dimensional orientation of cysteine ligands without introducing a CysXXCysXXCys motif. Tyr13 of AvFdI occupies the position of the fourth ligating cysteine in the homologous and structurally characterized 8Fe ferredoxin from Peptococcus aerogenes and a Y13C variant of AvFdI could be easily modeled as an 8Fe protein. However, characterization of purified Y13C FdI by UV-visible spectra, circular dichroism, electron paramagnetic resonance spectroscopies, and by x-ray crystallography revealed that the protein failed to use the introduced cysteine as a ligand and retained its [3Fe-4S]+/0 cluster. Further, electrochemical characterization showed that the redox potential and pH behavior 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 x-ray structure showed that the introduced cysteine was modified to become a persulfide. This modification may have occurred in vivo via the action of NifS, which is known to be expressed under 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 specificity to the reaction.
About this Structure
1FTC is a Single protein structure of sequence from Azotobacter vinelandii. Full crystallographic information is available from OCA.
Reference
Y13C Azotobacter vinelandii ferredoxin I. A designed [Fe-S] ligand motif contains a cysteine persulfide., Kemper MA, Stout CD, Lloyd SJ, Prasad GS, Fawcett SE, Armstrong FA, Shen B, Burgess BK, J Biol Chem. 1997 Jun 20;272(25):15620-7. PMID:9188450 Page seeded by OCA on Fri May 2 16:44:35 2008
