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

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

Regulation of Sulfur Assimilation Pathways in Burkholderia cenocepacia : Identification of Transcription Factors CysB and SsuR and Their Role in Control of Target Genes

“…Notably, a gene encoding an ortholog of CysC (an APS kinase producing 3Ј phosphoadenosine 5Ј phosphosulfate from APS) is absent from this gene cluster. However, there is an ORF encoding a putative ortholog of bacterial "CysH"-type reductases that utilizes APS as a substrate (1,5,26,28). The cluster also encodes a putative sulfite reductase component (CysI), which is similar (43%) to the hemoprotein subunit of E. coli sulfite reductase and 78% similar to that of P. aeruginosa (21), and a putative ortholog of uroporphyrinogen III methylase (CysG), an enzyme involved in synthesis of the siroheme cofactor of sulfite reductase (60).…”

Spectroscopic Studies on the [4Fe-4S] Cluster in Adenosine 5′-Phosphosulfate Reductase from Mycobacterium tuberculosis

“…2ϩ cluster in PaAPR is partially converted to the [3Fe-4S] ϩ form by treatment with the oxidant potassium ferricyanide (16). To assess this possibility for MtAPR, a stoichiometric amount of potassium ferricyanide was added to the enzyme.…”

“…Other interactions with the iron-sulfur cluster involve Thr-87 and Trp-246. In the active site, the phosphosulfate group of APS is positioned opposite the [4Fe-4S] cluster, and although no atoms intervene, the sulfate moiety is not in direct contact with the [4Fe-4S] cluster.Given the unusual Cys-Cys dyad coordination and its requirement for catalytic activity, defining the function and properties of the iron-sulfur cluster in APR has generated considerable interest (1,5,7,16,17). Most proteins containing [4Fe-4S] clusters are redox-active (18 -21); however, the [4Fe-4S] 2ϩ cluster in APR does not undergo redox changes during the catalytic cycle (1).…”

“…A purely structural role also appears unlikely in light of biophysical data obtained on the apo form of APR (6, 13, 16) and the fact that APR and PAPR share a common protein fold (5,11,12). Unfortunately, progress in this area has been hampered by the failure to generate a paramagnetic state of the [4Fe-4S] cluster that can be studied by EPR spectroscopy and related methods (16,17,22).Herein, we report the EPR spectra of MtAPR in the [4Fe-4S] ϩ state. The EPR spectrum of MtAPR displays a rhombic signal but is complex and consists of at least two overlapping S ϭ 1 ⁄ 2 species.…”

“…A purely structural role also appears unlikely in light of biophysical data obtained on the apo form of APR (6,13,16) and the fact that APR and PAPR share a common protein fold (5,11,12). Unfortunately, progress in this area has been hampered by the failure to generate a paramagnetic state of the [4Fe-4S] cluster that can be studied by EPR spectroscopy and related methods (16,17,22).…”