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 = ½ 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: Devayani P Bhave, Jiyoung A Hong, Michael Lee, Wei Jiang, Carsten Krebs, Kate S Carroll
• Journal: The Journal of biological chemistry (J Biol Chem) Vol. 286 Issue 2 Pg. 1216-26 (Jan 14 2011) ISSN: 1083-351X [Electronic] United States
• PMID: 21075841 (Publication Type: Journal Article)

Chemical References

• Ferricyanides
• Iron-Sulfur Proteins
• hexacyanoferrate III
• Oxidoreductases Acting on Sulfur Group Donors
• adenylylsulfate reductase

Topics

• Catalytic Domain
• Crystallization
• Drug Design
• Electron Spin Resonance Spectroscopy
• Ferricyanides (metabolism)
• Iron-Sulfur Proteins (chemistry, genetics, metabolism)
• Models, Chemical
• Mutagenesis, Site-Directed
• Mycobacterium tuberculosis (enzymology, genetics)
• Oxidation-Reduction
• Oxidoreductases Acting on Sulfur Group Donors (chemistry, genetics, metabolism)
• Protein Structure, Tertiary
• Substrate Specificity