Properties of the cysteine residues and iron-sulfur cluster of the assimilatory 5'-adenylyl sulfate reductase from Pseudomonas aeruginosa.
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.
Kim
Sung-Kun
SK
Department of Chemistry and Biochemistry, Texas Tech University, Lubbock, Texas 79409-1061, USA.
Rahman
Afroza
A
Bick
Julie-Ann
JA
Conover
Richard C
RC
Johnson
Michael K
MK
Mason
Jeremy T
JT
Hirasawa
Masakazu
M
Leustek
Thomas
T
Knaff
David B
DB
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification; D003545:Cysteine / Q000737:chemistry* / Q000235:genetics; D004578:Electron Spin Resonance Spectroscopy; D007506:Iron-Sulfur Proteins / Q000737:chemistry* / Q000235:genetics; D008024:Ligands; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010084:Oxidation-Reduction; D050862:Oxidoreductases Acting on Sulfur Group Donors / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification; D011550:Pseudomonas aeruginosa / Q000201:enzymology*; D011994:Recombinant Proteins / Q000737:chemistry; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013056:Spectrophotometry, Ultraviolet; D013059:Spectrum Analysis, Raman
D001426:Bacterial Proteins; D007506:Iron-Sulfur Proteins; D008024:Ligands; D011994:Recombinant Proteins; D050862:Oxidoreductases Acting on Sulfur Group Donors; C019618:adenylylsulfate reductase; D003545:Cysteine