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

Research output: Contribution to journalArticle

14 Citations (Scopus)

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 = 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
Volume286
Issue number2
DOIs
StatePublished - Jan 14 2011

Fingerprint

Mycobacterium tuberculosis
Sulfur
Paramagnetic resonance
Iron
Adenosine Phosphosulfate
Iron-Sulfur Proteins
Antitubercular Agents
Electron Spin Resonance Spectroscopy
Site-Directed Mutagenesis
Catalysis
Adenosine Diphosphate
Sulfates
Amino Acid Sequence
Catalytic Domain
Spectrum Analysis
Mutagenesis
Diphosphates
Ligands
Substrates
Ports and harbors

All Science Journal Classification (ASJC) codes

  • Biochemistry
  • Molecular Biology
  • Cell Biology

Cite this

Bhave, Devayani P. ; Hong, Jiyoung A. ; Lee, Michael ; Jiang, Wei ; Krebs, Carsten ; Carroll, Kate S. / Spectroscopic studies on the [4Fe-4S] cluster in adenosine 5′-phosphosulfate reductase from Mycobacterium tuberculosis. In: Journal of Biological Chemistry. 2011 ; Vol. 286, No. 2. pp. 1216-1226.
@article{dc79fb52344e4a63aca42f567e109764,
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 = 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.",
author = "Bhave, {Devayani P.} and Hong, {Jiyoung A.} and Michael Lee and Wei Jiang and Carsten Krebs and Carroll, {Kate S.}",
year = "2011",
month = "1",
day = "14",
doi = "10.1074/jbc.M110.193722",
language = "English (US)",
volume = "286",
pages = "1216--1226",
journal = "Journal of Biological Chemistry",
issn = "0021-9258",
publisher = "American Society for Biochemistry and Molecular Biology Inc.",
number = "2",

}

Spectroscopic studies on the [4Fe-4S] cluster in adenosine 5′-phosphosulfate reductase from Mycobacterium tuberculosis. / Bhave, Devayani P.; Hong, Jiyoung A.; Lee, Michael; Jiang, Wei; Krebs, Carsten; Carroll, Kate S.

In: Journal of Biological Chemistry, Vol. 286, No. 2, 14.01.2011, p. 1216-1226.

Research output: Contribution to journalArticle

TY - JOUR

T1 - Spectroscopic studies on the [4Fe-4S] cluster in adenosine 5′-phosphosulfate reductase from Mycobacterium tuberculosis

AU - Bhave, Devayani P.

AU - Hong, Jiyoung A.

AU - Lee, Michael

AU - Jiang, Wei

AU - Krebs, Carsten

AU - Carroll, Kate S.

PY - 2011/1/14

Y1 - 2011/1/14

N2 - 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.

AB - 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.

UR - http://www.scopus.com/inward/record.url?scp=78651390685&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=78651390685&partnerID=8YFLogxK

U2 - 10.1074/jbc.M110.193722

DO - 10.1074/jbc.M110.193722

M3 - Article

C2 - 21075841

AN - SCOPUS:78651390685

VL - 286

SP - 1216

EP - 1226

JO - Journal of Biological Chemistry

JF - Journal of Biological Chemistry

SN - 0021-9258

IS - 2

ER -