title: Spectroscopic studies on the [4Fe-4S] cluster in adenosine 5'-phosphosulfate reductase from Mycobacterium tuberculosis.
abstract: Mycobacterium tuberculosis adenosine 5'-phosphosulfate reductase (MtAPR) is an iron-sulfur protein and a validated target to develop new antitubercular agents, particularly for the treatment of latent infection. The enzyme harbors a [4Fe-4S](2+) cluster that is coordinated by four cysteinyl ligands, two of which are adjacent in the amino acid sequence. The iron-sulfur cluster is essential for catalysis; however, the precise role of the [4Fe-4S] cluster in APR remains unknown. 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 electron paramagnetic resonance spectroscopy. Herein, we overcome this limitation and report the EPR spectra of MtAPR in the [4Fe-4S](+) state. The EPR signal is rhombic and consists of two overlapping S = &frac12; species. Substrate binding to MtAPR led to a marked increase in the intensity and resolution of the EPR signal and to minor shifts in principle g values that were not observed among a panel of substrate analogs, including adenosine 5'-diphosphate. Using site-directed mutagenesis, in conjunction with kinetic and EPR studies, we have also identified an essential role for the active site residue Lys-144, whose side chain interacts with both the iron-sulfur cluster and the sulfate group of adenosine 5'-phosphosulfate. The implications of these findings are discussed with respect to the role of the iron-sulfur cluster in the catalytic mechanism of APR.
authors.affiliation: University of Michigan, Ann Arbor, Michigan 48109-2216, USA.
authors.forename: Devayani P
authors.initials: DP
authors.lastname: Bhave
authors.forename: Jiyoung A
authors.initials: JA
authors.lastname: Hong
authors.forename: Michael
authors.initials: M
authors.lastname: Lee
authors.forename: Wei
authors.initials: W
authors.lastname: Jiang
authors.forename: Carsten
authors.initials: C
authors.lastname: Krebs
authors.forename: Kate S
authors.initials: KS
authors.lastname: Carroll
mesh_terms: D020134:Catalytic Domain; D003460:Crystallization; D015195:Drug Design; D004578:Electron Spin Resonance Spectroscopy; D005292:Ferricyanides / Q000378:metabolism; D007506:Iron-Sulfur Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D008956:Models, Chemical; D016297:Mutagenesis, Site-Directed; D009169:Mycobacterium tuberculosis / Q000201:enzymology* / Q000235:genetics*; D010084:Oxidation-Reduction; D050862:Oxidoreductases Acting on Sulfur Group Donors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D017434:Protein Structure, Tertiary; D013379:Substrate Specificity
chemical_list: D005292:Ferricyanides; D007506:Iron-Sulfur Proteins; C007931:hexacyanoferrate III; D050862:Oxidoreductases Acting on Sulfur Group Donors; C019618:adenylylsulfate reductase