title: The interaction of 5'-adenylylsulfate reductase from Pseudomonas aeruginosa with its substrates.
abstract: APS reductase from Pseudomonas aeruginosa has been shown to form a disulfide-linked adduct with mono-cysteine variants of Escherichia coli thioredoxin and Chlamydomonas reinhardtii thioredoxin h1. These adducts presumably represent trapped versions of the intermediates formed during the catalytic cycle of this thioredoxin-dependent enzyme. The oxidation-reduction midpoint potential of the disulfide bond in the P. aeruginosa APS reductase/C. reinhardtii thioredoxin h1 adduct is -280 mV. Site-directed mutagenesis and mass spectrometry have identified Cys256 as the P. aeruginosa APS reductase residue that forms a disulfide bond with Cys36 of C. reinhardtii TRX h1 and Cys32 of E. coli thioredoxin in these adducts. Spectral perturbation measurements indicate that P. aeruginosa APS reductase can also form a non-covalent complex with E. coli thioredoxin and with C. reinhardtii thioredoxin h1. Perturbation of the resonance Raman and visible-region absorbance spectra of the APS reductase [4Fe-4S] center by either APS or the competitive inhibitor 5'-AMP indicates that both the substrate and product bind in close proximity to the cluster. These results have been interpreted in terms of a scheme in which one of the redox-active cysteine residues serves as the initial reductant for APS bound at or in close proximity to the [4Fe-4S] cluster.
authors.affiliation: Department of Chemistry and Biochemistry, Texas Tech University, Lubbock, Texas 79409-1061, USA. sung-kun.kim@ttu.edu
authors.forename: Sung-Kun
authors.initials: SK
authors.lastname: Kim
authors.forename: Afroza
authors.initials: A
authors.lastname: Rahman
authors.forename: Jeremy T
authors.initials: JT
authors.lastname: Mason
authors.forename: Masakazu
authors.initials: M
authors.lastname: Hirasawa
authors.forename: Richard C
authors.initials: RC
authors.lastname: Conover
authors.forename: Michael K
authors.initials: MK
authors.lastname: Johnson
authors.forename: Myroslawa
authors.initials: M
authors.lastname: Miginiac-Maslow
authors.forename: Eliane
authors.initials: E
authors.lastname: Keryer
authors.forename: David B
authors.initials: DB
authors.lastname: Knaff
authors.forename: Thomas
authors.initials: T
authors.lastname: Leustek
mesh_terms: D000250:Adenosine Phosphosulfate / Q000378:metabolism; D003545:Cysteine / Q000737:chemistry* / Q000235:genetics; D004220:Disulfides / Q000737:chemistry; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D010084:Oxidation-Reduction; D050862:Oxidoreductases Acting on Sulfur Group Donors / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D011550:Pseudomonas aeruginosa / Q000201:enzymology*; D013059:Spectrum Analysis, Raman; D013379:Substrate Specificity; D054480:Thioredoxin h; D013879:Thioredoxins / Q000737:chemistry*
chemical_list: D004220:Disulfides; D054480:Thioredoxin h; D000250:Adenosine Phosphosulfate; D013879:Thioredoxins; D050862:Oxidoreductases Acting on Sulfur Group Donors; C019618:adenylylsulfate reductase; D003545:Cysteine