title: Properties of the cysteine residues and iron-sulfur cluster of the assimilatory 5'-adenylyl sulfate reductase from Pseudomonas aeruginosa.
abstract: APS reductase from Pseudomonas aeruginosa has been shown to contain a [4Fe-4S] cluster. Thiol determinations and site-directed mutagenesis studies indicate that the single [4Fe-4S] cluster contains only three cysteine ligands, instead of the more typical arrangement in which clusters are bound to the protein by four cysteines. Resonance Raman studies in the Fe-S stretching region are also consistent with the presence of a redox-inert [4Fe-4S](2+) cluster with three cysteinate ligands and indicate that the fourth ligand is likely to be an oxygen-containing species. This conclusion is supported by resonance Raman and electron paramagnetic resonance (EPR) evidence for near stoichiometric conversion of the cluster to a [3Fe-4S](+) form by treatment with a 3-fold excess of ferricyanide. Site-directed mutagenesis experiments have identified Cys139, Cys228, and Cys231 as ligands to the cluster. The remaining two cysteines present in the enzyme, Cys140 and Cys256, form a redox-active disulfide/dithiol couple (E(m) = -300 mV at pH 7.0) that appears to play a role in the catalytic mechanism of the enzyme.
authors.affiliation: Department of Chemistry and Biochemistry, Texas Tech University, Lubbock, Texas 79409-1061, USA.
authors.forename: Sung-Kun
authors.initials: SK
authors.lastname: Kim
authors.forename: Afroza
authors.initials: A
authors.lastname: Rahman
authors.forename: Julie-Ann
authors.initials: JA
authors.lastname: Bick
authors.forename: Richard C
authors.initials: RC
authors.lastname: Conover
authors.forename: Michael K
authors.initials: MK
authors.lastname: Johnson
authors.forename: Jeremy T
authors.initials: JT
authors.lastname: Mason
authors.forename: Masakazu
authors.initials: M
authors.lastname: Hirasawa
authors.forename: Thomas
authors.initials: T
authors.lastname: Leustek
authors.forename: David B
authors.initials: DB
authors.lastname: Knaff
mesh_terms: D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification; D003545:Cysteine / Q000737:chemistry* / Q000235:genetics; D004578:Electron Spin Resonance Spectroscopy; D007506:Iron-Sulfur Proteins / Q000737:chemistry* / Q000235:genetics; D008024:Ligands; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010084:Oxidation-Reduction; D050862:Oxidoreductases Acting on Sulfur Group Donors / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification; D011550:Pseudomonas aeruginosa / Q000201:enzymology*; D011994:Recombinant Proteins / Q000737:chemistry; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013056:Spectrophotometry, Ultraviolet; D013059:Spectrum Analysis, Raman
chemical_list: D001426:Bacterial Proteins; D007506:Iron-Sulfur Proteins; D008024:Ligands; D011994:Recombinant Proteins; D050862:Oxidoreductases Acting on Sulfur Group Donors; C019618:adenylylsulfate reductase; D003545:Cysteine