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Title: Fluorescent Probes for Tracking the Transfer of Iron–Sulfur Cluster and Other Metal Cofactors in Biosynthetic Reaction Pathways

Abstract

Iron–sulfur (Fe–S) clusters are protein cofactors that are constructed and delivered to target proteins by elaborate biosynthetic machinery. Mechanistic insights into these processes have been limited by the lack of sensitive probes for tracking Fe–S cluster synthesis and transfer reactions. Here we present fusion protein- and intein-based fluorescent labeling strategies that can probe Fe–S cluster binding. The fluorescence is sensitive to different cluster types ([2Fe–2S] and [4Fe–4S] clusters), ligand environments ([2Fe–2S] clusters on Rieske, ferredoxin (Fdx), and glutaredoxin), and cluster oxidation states. The power of this approach is highlighted with an extreme example in which the kinetics of Fe–S cluster transfer reactions are monitored between two Fdx molecules that have identical Fe–S spectroscopic properties. This exchange reaction between labeled and unlabeled Fdx is catalyzed by dithiothreitol (DTT), a result that was confirmed by mass spectrometry. DTT likely functions in a ligand substitution reaction that generates a [2Fe–2S]–DTT species, which can transfer the cluster to either labeled or unlabeled Fdx. The ability to monitor this challenging cluster exchange reaction indicates that real-time Fe–S cluster incorporation can be tracked for a specific labeled protein in multicomponent assays that include several unlabeled Fe–S binding proteins or other chromophores. Such advanced kinetic experiments aremore » required to untangle the intricate networks of transfer pathways and the factors affecting flux through branch points. High sensitivity and suitability with high-throughput methodology are additional benefits of this approach. Lastly, we anticipate that this cluster detection methodology will transform the study of Fe–S cluster pathways and potentially other metal cofactor biosynthetic pathways.« less

Authors:
 [1];  [1];  [1];  [1];  [1];  [1]
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  1. Department of Biochemistry and Biophysics and ‡Department of Chemistry, Texas A&M University, College Station, Texas 77842-3012, United States
Publication Date:
Research Org.:
Texas A&M Univ., College Station, TX (United States)
Sponsoring Org.:
USDOE Office of Science (SC), Basic Energy Sciences (BES)
OSTI Identifier:
1167284
Alternate Identifier(s):
OSTI ID: 1346169
Grant/Contract Number:  
FG02-04ER15520
Resource Type:
Published Article
Journal Name:
Journal of the American Chemical Society
Additional Journal Information:
Journal Name: Journal of the American Chemical Society Journal Volume: 137 Journal Issue: 1; Journal ID: ISSN 0002-7863
Publisher:
American Chemical Society
Country of Publication:
United States
Language:
English
Subject:
37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CHEMISTRY

Citation Formats

Vranish, James N., Russell, William K., Yu, Lusa E., Cox, Rachael M., Russell, David H., and Barondeau, David P. Fluorescent Probes for Tracking the Transfer of Iron–Sulfur Cluster and Other Metal Cofactors in Biosynthetic Reaction Pathways. United States: N. p., 2014. Web. doi:10.1021/ja510998s.
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Vranish, James N., Russell, William K., Yu, Lusa E., Cox, Rachael M., Russell, David H., & Barondeau, David P. Fluorescent Probes for Tracking the Transfer of Iron–Sulfur Cluster and Other Metal Cofactors in Biosynthetic Reaction Pathways. United States. https://doi.org/10.1021/ja510998s
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Vranish, James N., Russell, William K., Yu, Lusa E., Cox, Rachael M., Russell, David H., and Barondeau, David P. Wed . "Fluorescent Probes for Tracking the Transfer of Iron–Sulfur Cluster and Other Metal Cofactors in Biosynthetic Reaction Pathways". United States. https://doi.org/10.1021/ja510998s.
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@article{osti_1167284,
title = {Fluorescent Probes for Tracking the Transfer of Iron–Sulfur Cluster and Other Metal Cofactors in Biosynthetic Reaction Pathways},
author = {Vranish, James N. and Russell, William K. and Yu, Lusa E. and Cox, Rachael M. and Russell, David H. and Barondeau, David P.},
abstractNote = {Iron–sulfur (Fe–S) clusters are protein cofactors that are constructed and delivered to target proteins by elaborate biosynthetic machinery. Mechanistic insights into these processes have been limited by the lack of sensitive probes for tracking Fe–S cluster synthesis and transfer reactions. Here we present fusion protein- and intein-based fluorescent labeling strategies that can probe Fe–S cluster binding. The fluorescence is sensitive to different cluster types ([2Fe–2S] and [4Fe–4S] clusters), ligand environments ([2Fe–2S] clusters on Rieske, ferredoxin (Fdx), and glutaredoxin), and cluster oxidation states. The power of this approach is highlighted with an extreme example in which the kinetics of Fe–S cluster transfer reactions are monitored between two Fdx molecules that have identical Fe–S spectroscopic properties. This exchange reaction between labeled and unlabeled Fdx is catalyzed by dithiothreitol (DTT), a result that was confirmed by mass spectrometry. DTT likely functions in a ligand substitution reaction that generates a [2Fe–2S]–DTT species, which can transfer the cluster to either labeled or unlabeled Fdx. The ability to monitor this challenging cluster exchange reaction indicates that real-time Fe–S cluster incorporation can be tracked for a specific labeled protein in multicomponent assays that include several unlabeled Fe–S binding proteins or other chromophores. Such advanced kinetic experiments are required to untangle the intricate networks of transfer pathways and the factors affecting flux through branch points. High sensitivity and suitability with high-throughput methodology are additional benefits of this approach. Lastly, we anticipate that this cluster detection methodology will transform the study of Fe–S cluster pathways and potentially other metal cofactor biosynthetic pathways.},
doi = {10.1021/ja510998s},
journal = {Journal of the American Chemical Society},
number = 1,
volume = 137,
place = {United States},
year = {Wed Dec 24 00:00:00 EST 2014},
month = {Wed Dec 24 00:00:00 EST 2014}
}
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Works referenced in this record:

Estimation of labile sulfide in iron-sulfur proteins
journal, October 1975

Building Fe–S proteins: bacterial strategies
journal, June 2010

  • Py, Béatrice; Barras, Frédéric
  • Nature Reviews Microbiology, Vol. 8, Issue 6
  • DOI: 10.1038/nrmicro2356

Ferrozine---a new spectrophotometric reagent for iron
journal, June 1970

  • Stookey, Lawrence L.
  • Analytical Chemistry, Vol. 42, Issue 7, p. 779-781
  • DOI: 10.1021/ac60289a016

Determination of quality of pyridoxal-5′-phosphate enzyme preparations by spectroscopic methods
journal, April 2005

  • Dršata, Jaroslav; Boušová, Iva; Maloň, Petr
  • Journal of Pharmaceutical and Biomedical Analysis, Vol. 37, Issue 5
  • DOI: 10.1016/j.jpba.2004.10.004

Structure, Function, and Formation of Biological Iron-Sulfur Clusters
journal, June 2005

Redox Control of Human Mitochondrial Outer Membrane Protein MitoNEET [2Fe-2S] Clusters by Biological Thiols and Hydrogen Peroxide
journal, January 2014

Physical and genetic map of the major nif gene cluster from Azotobacter vinelandii.
journal, February 1989

Molecular Mechanism of the Redox-dependent Interaction between NADH-dependent Ferredoxin Reductase and Rieske-type [2Fe-2S] Ferredoxin
journal, October 2007

  • Senda, Miki; Kishigami, Shinya; Kimura, Shigenobu
  • Journal of Molecular Biology, Vol. 373, Issue 2
  • DOI: 10.1016/j.jmb.2007.08.002

A Third Bacterial System for the Assembly of Iron-Sulfur Clusters with Homologs in Archaea and Plastids
journal, June 2002

  • Takahashi, Yasuhiro; Tokumoto, Umechiyo
  • Journal of Biological Chemistry, Vol. 277, Issue 32
  • DOI: 10.1074/jbc.C200365200

Spectroscopic and Functional Characterization of Iron-Bound Forms of Azotobacter vinelandii Nif IscA
journal, October 2012

  • Mapolelo, Daphne T.; Zhang, Bo; Naik, Sunil G.
  • Biochemistry, Vol. 51, Issue 41
  • DOI: 10.1021/bi300664j

The x-ray crystal structure of lysine-2,3-aminomutase from Clostridium subterminale
journal, September 2005

  • Lepore, B. W.; Ruzicka, F. J.; Frey, P. A.
  • Proceedings of the National Academy of Sciences, Vol. 102, Issue 39
  • DOI: 10.1073/pnas.0505726102

Studies on the Mechanism of Catalysis of Iron−Sulfur Cluster Transfer from IscU[2Fe2S] by HscA/HscB Chaperones
journal, November 2008

  • Bonomi, Francesco; Iametti, Stefania; Morleo, Anna
  • Biochemistry, Vol. 47, Issue 48
  • DOI: 10.1021/bi801565j

Redox Sensing: Novel Avenues and Paradigms
journal, April 2012

  • Ortiz de Orué Lucana, Darío
  • Antioxidants & Redox Signaling, Vol. 16, Issue 7
  • DOI: 10.1089/ars.2011.4466

Crystal Structure of Escherichia coli Fdx, an Adrenodoxin-Type Ferredoxin Involved in the Assembly of Iron−Sulfur Clusters
journal, August 2001

  • Kakuta, Yoshimitsu; Horio, Tatsuya; Takahashi, Yasuhiro
  • Biochemistry, Vol. 40, Issue 37
  • DOI: 10.1021/bi010544t

Metal trafficking: from maintaining the metal homeostasis to future drug design
journal, January 2009

  • Ba, Lalla Aicha; Doering, Mandy; Burkholz, Torsten
  • Metallomics, Vol. 1, Issue 4
  • DOI: 10.1039/b904533c

Maturation of Iron-Sulfur Proteins in Eukaryotes: Mechanisms, Connected Processes, and Diseases
journal, June 2008

Molecular organization, biochemical function, cellular role and evolution of NfuA, an atypical Fe-S carrier: Role of NfuA in Fe-S biosynthesis
journal, September 2012

Fluorescent indicators for Ca2+based on green fluorescent proteins and calmodulin
journal, August 1997

  • Miyawaki, Atsushi; Llopis, Juan; Heim, Roger
  • Nature, Vol. 388, Issue 6645
  • DOI: 10.1038/42264

Synthetic fluorescent sensors for studying the cell biology of metals
journal, February 2008

  • Domaille, Dylan W.; Que, Emily L.; Chang, Christopher J.
  • Nature Chemical Biology, Vol. 4, Issue 3
  • DOI: 10.1038/nchembio.69

Fluorescence Detection of a Protein-Bound 2Fe2S Cluster
journal, January 2009

Facilitated Transfer of IscU–[2Fe2S] Clusters by Chaperone-Mediated Ligand Exchange
journal, November 2011

  • Bonomi, Francesco; Iametti, Stefania; Morleo, Anna
  • Biochemistry, Vol. 50, Issue 44
  • DOI: 10.1021/bi201123z

Monothiol Glutaredoxins Function in Storing and Transporting [Fe 2 S 2 ] Clusters Assembled on IscU Scaffold Proteins
journal, September 2012

  • Shakamuri, Priyanka; Zhang, Bo; Johnson, Michael K.
  • Journal of the American Chemical Society, Vol. 134, Issue 37
  • DOI: 10.1021/ja306061x

Cochaperone Binding to LYR Motifs Confers Specificity of Iron Sulfur Cluster Delivery
journal, March 2014

Native Escherichia coli SufA, Coexpressed with SufBCDSE, Purifies as a [2Fe−2S] Protein and Acts as an Fe−S Transporter to Fe−S Target Enzymes
journal, April 2009

  • Gupta, Vibha; Sendra, Maïté; Naik, Sunil G.
  • Journal of the American Chemical Society, Vol. 131, Issue 17
  • DOI: 10.1021/ja807551e

Human Ind1, an Iron-Sulfur Cluster Assembly Factor for Respiratory Complex I
journal, November 2009

  • Sheftel, Alex D.; Stehling, Oliver; Pierik, Antonio J.
  • Molecular and Cellular Biology, Vol. 29, Issue 22
  • DOI: 10.1128/MCB.00817-09

Assembly of Iron-Sulfur Clusters: IDENTIFICATION OF AN
journal, May 1998

  • Zheng, Limin; Cash, Valerie L.; Flint, Dennis H.
  • Journal of Biological Chemistry, Vol. 273, Issue 21
  • DOI: 10.1074/jbc.273.21.13264

IscU as a Scaffold for Iron−Sulfur Cluster Biosynthesis:  Sequential Assembly of [2Fe-2S] and [4Fe-4S] Clusters in IscU †
journal, July 2000

  • Agar, Jeffrey N.; Krebs, Carsten; Frazzon, Jeverson
  • Biochemistry, Vol. 39, Issue 27
  • DOI: 10.1021/bi000931n

Fluorescence of a Histidine-Modified Enhanced Green Fluorescent Protein (EGFP) Effectively Quenched by Copper(II) Ions
journal, November 2012

  • Bálint, Emese-Éva; Petres, Judit; Szabó, Mária
  • Journal of Fluorescence, Vol. 23, Issue 2
  • DOI: 10.1007/s10895-012-1145-y

Reagent for the Detection of Protein Thiocarboxylates in the Bacterial Proteome: Lissamine Rhodamine B Sulfonyl Azide
journal, August 2010

  • Krishnamoorthy, Kalyanaraman; Begley, Tadhg P.
  • Journal of the American Chemical Society, Vol. 132, Issue 33
  • DOI: 10.1021/ja1034107

Specialized Function of Yeast Isa1 and Isa2 Proteins in the Maturation of Mitochondrial [4Fe-4S] Proteins
journal, December 2011

  • Mühlenhoff, Ulrich; Richter, Nadine; Pines, Ophry
  • Journal of Biological Chemistry, Vol. 286, Issue 48
  • DOI: 10.1074/jbc.M111.296152

In Vitro Activation of Apo-Aconitase Using a [4Fe-4S] Cluster-Loaded Form of the IscU [Fe−S] Cluster Scaffolding Protein †
journal, June 2007

  • Unciuleac, Mihaela-Carmen; Chandramouli, Kala; Naik, Sunil
  • Biochemistry, Vol. 46, Issue 23
  • DOI: 10.1021/bi6026665

Single-Molecule Analysis Reveals Differential Effect of ssDNA-Binding Proteins on DNA Translocation by XPD Helicase
journal, September 2009

Iron–sulfur cluster biogenesis and human disease
journal, August 2008

Iron–sulfur proteins in health and disease
journal, May 2010

  • Sheftel, Alex; Stehling, Oliver; Lill, Roland
  • Trends in Endocrinology & Metabolism, Vol. 21, Issue 5
  • DOI: 10.1016/j.tem.2009.12.006

Characteristics of self-quenching of the fluorescence of lipid-conjugated rhodamine in membranes.
journal, August 1990

The role of mitochondria in cellular iron–sulfur protein biogenesis and iron metabolism
journal, September 2012

  • Lill, Roland; Hoffmann, Bastian; Molik, Sabine
  • Biochimica et Biophysica Acta (BBA) - Molecular Cell Research, Vol. 1823, Issue 9
  • DOI: 10.1016/j.bbamcr.2012.05.009

A Proposed Role for the Azotobacter vinelandii NfuA Protein as an Intermediate Iron-Sulfur Cluster Carrier
journal, May 2008

  • Bandyopadhyay, Sibali; Naik, Sunil G.; O'Carroll, Ina P.
  • Journal of Biological Chemistry, Vol. 283, Issue 20
  • DOI: 10.1074/jbc.M709161200

Mechanism of glutaredoxin—ISU [2Fe–2S] cluster exchange
journal, January 2011

  • Qi, Wenbin; Cowan, J. A.
  • Chemical Communications, Vol. 47, Issue 17
  • DOI: 10.1039/c0cc05079b

Inborn errors of copper metabolism
book, April 2013

Two Rapid Catalyst-Free Click Reactions for In Vivo Protein Labeling of Genetically Encoded Strained Alkene/Alkyne Functionalities
journal, August 2014

  • Kurra, Yadagiri; Odoi, Keturah A.; Lee, Yan-Jiun
  • Bioconjugate Chemistry, Vol. 25, Issue 9
  • DOI: 10.1021/bc500361d

Glutathione Complexed Fe–S Centers
journal, June 2012

  • Qi, Wenbin; Li, Jingwei; Chain, C. Y.
  • Journal of the American Chemical Society, Vol. 134, Issue 26
  • DOI: 10.1021/ja302186j

Subcellular compartmentalization of human Nfu, an iron-sulfur cluster scaffold protein, and its ability to assemble a [4Fe-4S] cluster
journal, July 2003

  • Tong, W. -H.; Jameson, G. N. L.; Huynh, B. H.
  • Proceedings of the National Academy of Sciences, Vol. 100, Issue 17
  • DOI: 10.1073/pnas.1732541100

Structural Basis for Delivery of the Intact [Fe2S2] Cluster by Monothiol Glutaredoxin
journal, June 2009

  • Iwema, Thomas; Picciocchi, Antoine; Traore, Daouda A. K.
  • Biochemistry, Vol. 48, Issue 26
  • DOI: 10.1021/bi900440m

Mechanism and energetics of green fluorescent protein chromophore synthesis revealed by trapped intermediate structures
journal, October 2003

  • Barondeau, D. P.; Putnam, C. D.; Kassmann, C. J.
  • Proceedings of the National Academy of Sciences, Vol. 100, Issue 21
  • DOI: 10.1073/pnas.2133463100

IscA, an Alternate Scaffold for Fe−S Cluster Biosynthesis
journal, October 2001

  • Krebs, Carsten; Agar, Jeffrey N.; Smith, Archer D.
  • Biochemistry, Vol. 40, Issue 46
  • DOI: 10.1021/bi015656z

Extrusion of iron-sulfide (Fe2S2* and Fe4S4*) cores from the active sites of ferredoxin proteins
journal, January 1975

  • Que, L.; Holm, R. H.; Mortenson, L. E.
  • Journal of the American Chemical Society, Vol. 97, Issue 2
  • DOI: 10.1021/ja00835a064

Charting the travels of copper in eukaryotes from yeast to mammals
journal, September 2012

  • Nevitt, Tracy; Öhrvik, Helena; Thiele, Dennis J.
  • Biochimica et Biophysica Acta (BBA) - Molecular Cell Research, Vol. 1823, Issue 9
  • DOI: 10.1016/j.bbamcr.2012.02.011

HscA and HscB Stimulate [2Fe-2S] Cluster Transfer from IscU to Apoferredoxin in an ATP-Dependent Reaction †
journal, September 2006

  • Chandramouli, Kala; Johnson, Michael K.
  • Biochemistry, Vol. 45, Issue 37
  • DOI: 10.1021/bi061237w

Monothiol glutaredoxins and A-type proteins: partners in Fe–S cluster trafficking
journal, January 2013

  • Mapolelo, Daphne T.; Zhang, Bo; Randeniya, Sajini
  • Dalton Transactions, Vol. 42, Issue 9
  • DOI: 10.1039/c2dt32263c
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