PDBsum entry 1ftc

Electron transport

PDB id: 1ftc

Contents

Protein chains

106 a.a. *

Ligands

SF4 ×2

F3S ×2

* Residue conservation analysis

PDB id:

1ftc

Name:

Electron transport

Title:

Y13c mutant of azotobacter vinelandii fdi

Structure:

Ferredoxin. Chain: a, b. Engineered: yes. Mutation: yes. Other_details: cys 13 modified to a persulfide, [3fe-4s] and [4fe-4s] clusters as in native protein

Source:

Azotobacter vinelandii. Organism_taxid: 354. Strain: jg100. Expressed in: azotobacter vinelandii. Expression_system_taxid: 354

Resolution:

2.35Å R-factor: 0.216

Authors:

M.A.Kemper,S.J.Lloyd,G.S.Prasad,C.D.Stout,S.Fawcett,F.A.Armstrong, B.K.Burgess

Key ref:

M.A.Kemper et al. (1997). Y13C Azotobacter vinelandii ferredoxin I. A designed [Fe-S] ligand motif contains a cysteine persulfide. J Biol Chem, 272, 15620-15627. PubMed id: 9188450 DOI: 10.1074/jbc.272.25.15620

Date:

08-Jan-97 Release date: 01-Apr-97

Protein chains

P00214  (FER1_AZOVI) -  Ferredoxin-1 from Azotobacter vinelandii

Seq: 107 a.a.

Struc: 106 a.a.*

* PDB and UniProt seqs differ at 1 residue position (black cross)

DOI no: 10.1074/jbc.272.25.15620

J Biol Chem 272:15620-15627 (1997)

PubMed id: 9188450

Y13C Azotobacter vinelandii ferredoxin I. A designed [Fe-S] ligand motif contains a cysteine persulfide.

M.A.Kemper, C.D.Stout, S.J.Lloyd, G.S.Prasad, S.E.Fawcett, F.A.Armstrong, B.Shen, B.K.Burgess, S.E.Lloyd, S.Fawcett.

ABSTRACT

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.

Selected figure(s)

Figure 1.
Fig. 1. The structure of a CysXXCysXXCys loop in P. aerogenes 8Fe ferredoxin (PaFd), the structure of the corresponding CysXXCysXXXXCys loop in A. vinelandii 7Fe ferredoxin (AvFdI), and a model for the Y13C mutant of FdI (see text). Only side chains of Cys residues and Tyr13 of AvFdI are shown in addition to the [Fe-S] clusters. Carbon, nitrogen, oxygen, sulfur, and iron atoms are shaded white, light gray, gray, dark gray, and black, respectively.

Figure 3.
Fig. 3. Stereo figure of the protein environment of the CSS residue in the structure of Y13C FdI (thick lines) superimposed by least squares fit onto the structure of native FdI (thin lines). In this view the loop of the residues Cys8XXCys11X(13)XX Cys16 folds around the upper half of the [3Fe-4S] cluster while the^ loop of residues Pro47XCys49Pro50XXXIle^54 folds around the lower half of the cluster. The short contact between S of CSS13 and an inorganic S of the [3Fe-4S] cluster is indicated. Note the proximity of Pro50 and Pro87 on either side of residue 13. The surface of the protein is toward^ the right. Molecule A of Y13C FdI is shown; molecule B is very similar.

The above figures are reprinted by permission from the ASBMB: J Biol Chem (1997, 272, 15620-15627) copyright 1997.

Figures were selected by an automated process.