Properties of the Cysteine Residues and Iron−Sulfur Cluster of the Assimilatory 5‘-Adenylyl Sulfate Reductase from Pseudomonas aeruginosa

• Published in: Biochemistry (Easton) 2004-10, Vol.43 (42), p.13478-13486
• Main Authors: Kim, Sung-Kun, Rahman, Afroza, Bick, Julie-Ann, Conover, Richard C, Johnson, Michael K, Mason, Jeremy T, Hirasawa, Masakazu, Leustek, Thomas, Knaff, David B
• Format: Article
• Language: English
• Subjects: Amino Acid Sequence; Bacterial Proteins - chemistry; Bacterial Proteins - genetics; Bacterial Proteins - isolation & purification; Cysteine - chemistry; Cysteine - genetics; Electron Spin Resonance Spectroscopy; Iron-Sulfur Proteins - chemistry; Iron-Sulfur Proteins - genetics; Ligands; Molecular Sequence Data; Mutagenesis, Site-Directed; Oxidation-Reduction; Oxidoreductases Acting on Sulfur Group Donors - chemistry; Oxidoreductases Acting on Sulfur Group Donors - genetics; Oxidoreductases Acting on Sulfur Group Donors - isolation & purification; Pseudomonas aeruginosa; Pseudomonas aeruginosa - enzymology; Recombinant Proteins - chemistry; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; Spectrophotometry, Ultraviolet; Spectrum Analysis, Raman
• ISSN: 0006-2960; 1520-4995
• DOI: 10.1021/bi048811t

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 (E m = −300 mV at pH 7.0) that appears to play a role in the catalytic mechanism of the enzyme.