Spectroscopic studies on the [4Fe-4S] cluster in adenosine 5′-phosphosulfate reductase from Mycobacterium tuberculosis

Devayani P. Bhave, Jiyoung A. Hong, Michael Lee, Wei Jiang, Carsten Krebs, Kate S. Carroll

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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 = 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.

Original languageEnglish (US)
Pages (from-to)1216-1226
Number of pages11
JournalJournal of Biological Chemistry
Issue number2
StatePublished - Jan 14 2011

All Science Journal Classification (ASJC) codes

  • Biochemistry
  • Molecular Biology
  • Cell Biology


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