The interaction of 5'-adenylylsulfate reductase from Pseudomonas aeruginosa with its substrates |
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Authors: | Kim Sung-Kun Rahman Afroza Mason Jeremy T Hirasawa Masakazu Conover Richard C Johnson Michael K Miginiac-Maslow Myroslawa Keryer Eliane Knaff David B Leustek Thomas |
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Institution: | Department of Chemistry and Biochemistry, Texas Tech University, Lubbock, Texas 79409-1061, USA. sung-kun.kim@ttu.edu |
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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. |
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