title: Engineering a three-cysteine, one-histidine ligand environment into a new hyperthermophilic archaeal Rieske-type [2Fe-2S] ferredoxin from Sulfolobus solfataricus.
abstract: We heterologously overproduced a hyperthermostable archaeal low potential (E(m) = -62 mV) Rieske-type ferredoxin (ARF) from Sulfolobus solfataricus strain P-1 and its variants in Escherichia coli to examine the influence of ligand substitutions on the properties of the [2Fe-2S] cluster. While two cysteine ligand residues (Cys(42) and Cys(61)) are essential for the cluster assembly and/or stability, the contributions of the two histidine ligands to the cluster assembly in the archaeal Rieske-type ferredoxin appear to be inequivalent as indicated by much higher stability of the His(64) --> Cys variant (H64C) than the His(44) --> Cys variant (H44C). The x-ray absorption and resonance Raman spectra of the H64C variant firmly established the formation of a novel, oxidized [2Fe-2S] cluster with one histidine and three cysteine ligands in the archaeal Rieske-type protein moiety. Comparative resonance Raman features of the wild-type, natural abundance and uniformly (15)N-labeled ARF and its H64C variant showed significant mixing of the Fe-S and Fe-N stretching characters for an oxidized biological [2Fe-2S] cluster with partial histidine ligation.
authors.affiliation: Department of Biochemistry and Molecular Biology, Nippon Medical School, Sendagi, Bunkyo-ku, Tokyo 113-8602, Japan.
authors.forename: Asako
authors.initials: A
authors.lastname: Kounosu
authors.forename: Zhongrui
authors.initials: Z
authors.lastname: Li
authors.forename: Nathaniel J
authors.initials: NJ
authors.lastname: Cosper
authors.forename: Jacob E
authors.initials: JE
authors.lastname: Shokes
authors.forename: Robert A
authors.initials: RA
authors.lastname: Scott
authors.forename: Takeo
authors.initials: T
authors.lastname: Imai
authors.forename: Akio
authors.initials: A
authors.lastname: Urushiyama
authors.forename: Toshio
authors.initials: T
authors.lastname: Iwasaki
mesh_terms: D000595:Amino Acid Sequence; D001665:Binding Sites; D002942:Circular Dichroism; D003545:Cysteine / Q000737:chemistry*; D014450:Electron Transport Complex III / Q000737:chemistry*; D004926:Escherichia coli / Q000378:metabolism; D005288:Ferredoxins / Q000737:chemistry*; D006639:Histidine / Q000737:chemistry*; D007506:Iron-Sulfur Proteins / Q000737:chemistry*; D008024:Ligands; D008280:Magnetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010100:Oxygen / Q000378:metabolism; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000737:chemistry; D017386:Sequence Homology, Amino Acid; D013052:Spectrometry, X-Ray Emission; D013059:Spectrum Analysis, Raman; D016944:Sulfolobus / Q000378:metabolism*; D013696:Temperature; D013816:Thermodynamics; D014466:Ultraviolet Rays
chemical_list: D005288:Ferredoxins; D007506:Iron-Sulfur Proteins; D008024:Ligands; D011994:Recombinant Proteins; C037989:Rieske iron-sulfur protein; D006639:Histidine; D014450:Electron Transport Complex III; D003545:Cysteine; D010100:Oxygen