title: Crystal structures of the all-cysteinyl-coordinated D14C variant of Pyrococcus furiosus ferredoxin: [4Fe-4S] ↔ [3Fe-4S] cluster conversion.
abstract: The structure of the all-cysteinyl-coordinated D14C variant of [4Fe-4S] ferredoxin from the hyperthermophilic archaeon Pyrococcus furiosus has been determined to 1.7 Å resolution from a crystal belonging to space group C222(1) with two types of molecules, A and B, in the asymmetric unit. A and B molecules have different crystal packing and intramolecular disulfide bond conformation. The crystal packing reveals a β-sheet interaction between A molecules in adjacent asymmetric units, whereas B molecules are packed as monomers in a less rigid position next to the A-A extended β-sheet dimers. The A molecules contain an intramolecular disulfide bond in a double conformation with 60% occupancy left-handed and 40% occupancy right-handed spiral conformation, whereas B molecules have an intramolecular disulfide bond in a right-handed spiral conformation. The cluster in D14C [4Fe-4S] P. furiosus ferredoxin was chemically oxidized at pH 5.8 to [3Fe-4S]. For purification at pH 8.0, two forms of the protein are obtained. Mass spectrometric analysis shows that the two forms are the D14C [3Fe-4S] P. furiosus ferredoxin monomer and a disulfide-bonded dimer of D14C [3Fe-4S] P. furiosus ferredoxin. When oxidization and purification are carried out at pH 5.8, only the monomer is obtained. The crystal structure of D14C [3Fe-4S] P. furiosus ferredoxin monomer was determined to 2.8 Å resolution from a crystal belonging to space group P2(1)2(1)2(1) with two molecules in the asymmetric unit. The molecules resemble molecule A of D14C [4Fe-4S] P. furiosus ferredoxin and electron density clearly shows the presence of a [3Fe-4S] cluster.
authors.affiliation: Department of Chemistry, Technical University of Denmark.
authors.forename: Monika Nøhr
authors.initials: MN
authors.lastname: Løvgreen
authors.forename: Maja
authors.initials: M
authors.lastname: Martic
authors.forename: Michael S
authors.initials: MS
authors.lastname: Windahl
authors.forename: Hans E M
authors.initials: HE
authors.lastname: Christensen
authors.forename: Pernille
authors.initials: P
authors.lastname: Harris
mesh_terms: D000595:Amino Acid Sequence; D018360:Crystallography, X-Ray; D003545:Cysteine / Q000235:genetics; D005288:Ferredoxins / Q000737:chemistry* / Q000235:genetics* / Q000302:isolation & purification; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009154:Mutation; D011487:Protein Conformation; D020027:Pyrococcus furiosus / Q000737:chemistry* / Q000235:genetics; D016415:Sequence Alignment
chemical_list: D005288:Ferredoxins; D003545:Cysteine