1ftc
From Proteopedia
(New page: 200px<br /><applet load="1ftc" size="450" color="white" frame="true" align="right" spinBox="true" caption="1ftc, resolution 2.35Å" /> '''Y13C MUTANT OF AZOTO...) |
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| - | [[Image:1ftc.jpg|left|200px]]<br /><applet load="1ftc" size=" | + | [[Image:1ftc.jpg|left|200px]]<br /><applet load="1ftc" size="350" color="white" frame="true" align="right" spinBox="true" |
caption="1ftc, resolution 2.35Å" /> | caption="1ftc, resolution 2.35Å" /> | ||
'''Y13C MUTANT OF AZOTOBACTER VINELANDII FDI'''<br /> | '''Y13C MUTANT OF AZOTOBACTER VINELANDII FDI'''<br /> | ||
==Overview== | ==Overview== | ||
| - | Ferredoxins that contain [4Fe-4S]2+/+ clusters often obtain three of their | + | 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== | ==About this Structure== | ||
| - | 1FTC is a [http://en.wikipedia.org/wiki/Single_protein Single protein] structure of sequence from [http://en.wikipedia.org/wiki/Azotobacter_vinelandii Azotobacter vinelandii] with SF4 and F3S as [http://en.wikipedia.org/wiki/ligands ligands]. Full crystallographic information is available from [http:// | + | 1FTC is a [http://en.wikipedia.org/wiki/Single_protein Single protein] structure of sequence from [http://en.wikipedia.org/wiki/Azotobacter_vinelandii Azotobacter vinelandii] with <scene name='pdbligand=SF4:'>SF4</scene> and <scene name='pdbligand=F3S:'>F3S</scene> as [http://en.wikipedia.org/wiki/ligands ligands]. Full crystallographic information is available from [http://oca.weizmann.ac.il/oca-bin/ocashort?id=1FTC OCA]. |
==Reference== | ==Reference== | ||
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[[Category: Azotobacter vinelandii]] | [[Category: Azotobacter vinelandii]] | ||
[[Category: Single protein]] | [[Category: Single protein]] | ||
| - | [[Category: Armstrong, F | + | [[Category: Armstrong, F A.]] |
| - | [[Category: Burgess, B | + | [[Category: Burgess, B K.]] |
[[Category: Fawcett, S.]] | [[Category: Fawcett, S.]] | ||
| - | [[Category: Kemper, M | + | [[Category: Kemper, M A.]] |
| - | [[Category: Lloyd, S | + | [[Category: Lloyd, S J.]] |
| - | [[Category: Prasad, G | + | [[Category: Prasad, G S.]] |
| - | [[Category: Stout, C | + | [[Category: Stout, C D.]] |
[[Category: F3S]] | [[Category: F3S]] | ||
[[Category: SF4]] | [[Category: SF4]] | ||
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[[Category: iron-sulfur]] | [[Category: iron-sulfur]] | ||
| - | ''Page seeded by [http:// | + | ''Page seeded by [http://oca.weizmann.ac.il/oca OCA ] on Thu Feb 21 12:42:24 2008'' |
Revision as of 10:42, 21 February 2008
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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 with and as ligands. 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
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