The interaction of 5′-adenylylsulfate reductase from Pseudomonas aeruginosa with its substrates

Sung Kun Kim, Afroza Rahman, Jeremy T. Mason, Masakazu Hirasawa, Richard C. Conover, Michael K. Johnson, Myroslawa Miginiac-Maslow, Eliane Keryer, David B. Knaff, Thomas Leustek

Research output: Contribution to journalArticlepeer-review

15 Scopus citations

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.

Original languageEnglish (US)
Pages (from-to)103-112
Number of pages10
JournalBiochimica et Biophysica Acta - Bioenergetics
Volume1710
Issue number2-3
DOIs
StatePublished - Dec 20 2005

All Science Journal Classification (ASJC) codes

  • Biophysics
  • Biochemistry
  • Cell Biology

Keywords

  • Disulfide-linked protein complex
  • Iron-sulfur cluster
  • Pseudomonas aeruginosa APS reductase
  • Thioredoxin

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