Properties of the cysteine residues and iron-sulfur cluster of the assimilatory 5′-adenylyl sulfate reductase from Pseudomonas aeruginosa

Sung Kun Kim, Afroza Rahman, Julie Ann Bick, Richard C. Conover, Michael K. Johnson, Jeremy T. Mason, Masakazu Hirasawa, Thomas Leustek, David B. Knaff

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32 Scopus citations

Abstract

APS reductase from Pseudomonas aeruginosa has been shown to contain a [4Fe-4S] cluster. Thiol determinations and site-directed mutagenesis studies indicate that the single [4Fe-4S] cluster contains only three cysteine ligands, instead of the more typical arrangement in which clusters are bound to the protein by four cysteines. Resonance Raman studies in the Fe-S stretching region are also consistent with the presence of a redox-inert [4Fe-4S]2+ cluster with three cysteinate ligands and indicate that the fourth ligand is likely to be an oxygen-containing species. This conclusion is supported by resonance Raman and electron paramagnetic resonance (EPR) evidence for near stoichiometric conversion of the cluster to a [3Fe-4S]+ form by treatment with a 3-fold excess of ferricyanide. Site-directed mutagenesis experiments have identified Cys139, Cys228, and Cys231 as ligands to the cluster. The remaining two cysteines present in the enzyme, Cys140 and Cys256, form a redox-active disulfide/dithiol couple (Em = -300 mV at pH 7.0) that appears to play a role in the catalytic mechanism of the enzyme. 1.

Original languageEnglish (US)
Pages (from-to)13478-13486
Number of pages9
JournalBiochemistry
Volume43
Issue number42
DOIs
StatePublished - Oct 26 2004

All Science Journal Classification (ASJC) codes

  • Biochemistry

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