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Interactions of 5′‐Adenylylphosphosulfate Reductase from Pseudomonas aeruginosa with substrates
Author(s) -
Doffing Kimberly,
Shin Ben C.,
Leustek Thomas,
Knaff David,
Kim SungKun
Publication year - 2008
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.22.2_supplement.341
Subject(s) - chemistry , thioredoxin reductase , ferredoxin thioredoxin reductase , cysteine , thioredoxin , escherichia coli , adduct , chlamydomonas reinhardtii , stereochemistry , biochemistry , active site , resonance raman spectroscopy , enzyme , organic chemistry , raman spectroscopy , physics , mutant , optics , gene
5′‐Adenylylphosphosulfate (APS) reductase from a bacterium Pseudomonas aeruginosa (PaAPR) has been shown to contain a [4Fe‐4S] cluster. Thioredoxin is used as an electron donor for PaAPR has been shown to form a disulfide‐linked adduct with mono‐cysteine variants of Escherichia coli thioredoxin and Chlamydomonas reinhardtii thioredoxin‐dependent enzyme. The redox midpoint potential of the disulfide bond in the PaAPR and the mono‐cysteine adduct is −280mV. Site‐directed mutagenesis and mass spectrometry have identified Cys256 as the PaAPR that forms a disulfide bond with Cys36 of C. reinhardtii thioredoxin and Cys32 of E. coli thioredoxin in these adducts. 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. We also found that randomly generated single‐stranded deoxyribonucleic acid (ssDNA) seems to bind PaAPR in the presence of [4Fe‐4S] cluster using an electrophoretic mobility shift assay technique, suggesting that the phosphate moiety of the random pool of ssDNA can be a binding site of APS reductase.