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 ironsulfur 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 ؍ 1 ⁄ 2 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.In bacteria and plants, activation of inorganic sulfur is required for de novo biosynthesis of cysteine. To this end, the metabolic assimilation of sulfate from the environment proceeds via adenosine 5Ј-phosphosulfate (APS) 2 or 3Ј-phosphoadenosine-5Ј-phosphosulfate (PAPS) (1). These intermediates are produced by the action of ATP-sulfurylase (EC 2.7.7.4), which condenses sulfate and ATP to form APS (2, 3), and by APS kinase (EC 2.7.1.25), which produces PAPS from ATP and APS (4).APS and PAPS are reduced by enzymes in the reductive branch of the sulfate assimilation pathway, producing sulfite and AMP or adenosine 3Ј,5Ј-diphosphate (Scheme 1). These enzymes can be subdivided into two groups according to their substrate preference: the APS reductases (APR) and the PAPS reductases (PAPR) (EC 1.8.99.4). Functional and structural studies have been used to investigate the chemical reaction mechanism of APR and PAPR enzymes (1,(5)(6)(7)(8). The mechanism involves nucleophilic attack by the active site cysteine on the sulfur atom of APS or PAPS to form an enzyme S-sulfocysteine intermediate, which is cleaved by thiol-disulfide exchange with thioredoxin or glutaredoxin (Fig. 1). The sulfite product is then reduced to sulfide by sulfite reductase (EC 1.8.7.1) and utilized to synthesize cysteine and other essential sulfur-containing biomolecules (9). In the human pathogen Mycobacterium tuberculosis, APR is a validated target against the latent phase of infection (10).Only a 3Ј-phosphate group distinguishes PAPS from APS. Accordingly, APR and PAPR have nearly identical three...