title: Heterometallic [AgFe(3)S          (4)] ferredoxin variants: synthesis, characterization, and the first crystal structure of an engineered heterometallic iron-sulfur protein.
abstract: Heterometallic [AgFe(3)S(4)] iron-sulfur clusters assembled in wild-type Pyrococcus furiosus ferredoxin and two variants, D14C and D14H, are characterized. The crystal structure of the [AgFe(3)S(4)] D14C variant shows that the silver(I) ion is indeed part of the cluster and is coordinated to the thiolate group of residue 14. Cyclic voltammetry shows one redox pair with a reduction potential of +220 mV versus the standard hydrogen electrode which is assigned to the [AgFe(3)S(4)](2+/+) couple. The oxidized form of the [AgFe(3)S(4)] D14C variant is stable in the presence of dioxygen, whereas the oxidized forms of the [AgFe(3)S(4)] wild type and D14H variants convert to the [Fe(3)S(4)] ferredoxin form. The monovalent d (10) silver(I) ion stabilizes the [Fe(3)S(4)](+/0) cluster fragment, as opposed to divalent d (10) metal ions, resulting in more than 0.4 V difference in reduction potentials between the silver(I) and, e.g., zinc(II) heterometallic [MFe(3)S(4)] ferredoxins. The trend in reduction potentials for the variants containing the [AgFe(3)S(4)] cluster is wild type ≤ D14C < D14H and shows the same trend as reported for the variants containing the [Fe(3)S(4)] cluster, but is different from the D14C < D14H < wild type trend reported for the [Fe(4)S(4)] ferredoxin. The similarity in the reduction potential trend for the variants containing the heterometallic [AgFe(3)S(4)] cluster and the [Fe(3)S(4)] cluster can be rationalized in terms of the electrostatic influence of the residue 14 side chains, rather than the dissociation constant of this residue, as is the case for [Fe(4)S(4)] ferredoxins. The trends in reduction potentials are in line with there being no electronic coupling between the silver(I) ion and the Fe(3)S(4) fragment.
authors.affiliation: Department of Chemistry, Technical University of Denmark, Kemitorvet 207, 2800, Kongens Lyngby, Denmark.
authors.forename: Maja
authors.initials: M
authors.lastname: Martic
authors.affiliation: Department of Chemistry, Technical University of Denmark, Kemitorvet 207, 2800, Kongens Lyngby, Denmark.
authors.forename: Ida Noémi
authors.initials: IN
authors.lastname: Jakab-Simon
authors.affiliation: Department of Chemistry, Technical University of Denmark, Kemitorvet 207, 2800, Kongens Lyngby, Denmark.
authors.forename: Lærke Tvedebrink
authors.initials: LT
authors.lastname: Haahr
authors.affiliation: Department of Biotechnology, Delft University of Technology, Julianalaan 67, 2628 BC, Delft, The Netherlands.
authors.forename: Wilfred Raymond
authors.initials: WR
authors.lastname: Hagen
authors.affiliation: Department of Chemistry, Technical University of Denmark, Kemitorvet 207, 2800, Kongens Lyngby, Denmark. hemc@kemi.dtu.dk.
authors.forename: Hans Erik Mølager
authors.initials: HEM
authors.lastname: Christensen
mesh_terms: D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D016880:Anisotropy; D019843:Archaeal Proteins / Q000096:biosynthesis / Q000737:chemistry* / Q000235:genetics; D056831:Coordination Complexes / Q000737:chemistry*; D018360:Crystallography, X-Ray; D004578:Electron Spin Resonance Spectroscopy; D004926:Escherichia coli; D005288:Ferredoxins / Q000096:biosynthesis / Q000737:chemistry* / Q000235:genetics; D007501:Iron / Q000737:chemistry*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010084:Oxidation-Reduction; D015202:Protein Engineering; D055550:Protein Stability; D020027:Pyrococcus furiosus; D012834:Silver / Q000737:chemistry*
chemical_list: D019843:Archaeal Proteins; D056831:Coordination Complexes; D005288:Ferredoxins; D012834:Silver; D007501:Iron