The interaction of 5'-adenylylsulfate reductase from Pseudomonas aeruginosa with its substrates.
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.
Kim
Sung-Kun
SK
Department of Chemistry and Biochemistry, Texas Tech University, Lubbock, Texas 79409-1061, USA. sung-kun.kim@ttu.edu
Rahman
Afroza
A
Mason
Jeremy T
JT
Hirasawa
Masakazu
M
Conover
Richard C
RC
Johnson
Michael K
MK
Miginiac-Maslow
Myroslawa
M
Keryer
Eliane
E
Knaff
David B
DB
Leustek
Thomas
T
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*
D004220:Disulfides; D054480:Thioredoxin h; D000250:Adenosine Phosphosulfate; D013879:Thioredoxins; D050862:Oxidoreductases Acting on Sulfur Group Donors; C019618:adenylylsulfate reductase; D003545:Cysteine