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Title: The N-terminal Domain of Escherichia coli Assimilatory NADPH-Sulfite Reductase Hemoprotein Is an Oligomerization Domain That Mediates Holoenzyme Assembly

Abstract

Assimilatory NADPH-sulfite reductase (SiR) from Escherichia coli is a structurally complex oxidoreductase that catalyzes the six-electron reduction of sulfite to sulfide. Two subunits, one a flavin-binding flavoprotein (SiRFP, the α subunit) and the other an iron-containing hemoprotein (SiRHP, the β subunit), assemble to make a holoenzyme of about 800 kDa. How the two subunits assemble is not known. The iron-rich cofactors in SiRHP are unique because they are a covalent arrangement of a Fe 4S 4 cluster attached through a cysteine ligand to an iron-containing porphyrinoid called siroheme. The link between cofactor biogenesis and SiR stability is also ill-defined. By use of hydrogen/deuterium exchange and biochemical analysis, we show that the α 8β 4 SiR holoenzyme assembles through the N terminus of SiRHP and the NADPH binding domain of SiRFP. By use of small angle x-ray scattering, we explore the structure of the SiRHP N-terminal oligomerization domain. We additionally report a novel form of the hemoprotein that occurs in the absence of its cofactors. Apo-SiRHP forms a homotetramer, also dependent on its N terminus, that is unable to assemble with SiRFP. Finally, from these results, we propose that homotetramerization of apo-SiRHP serves as a quality control mechanism to prevent formationmore » of inactive holoenzyme in the case of limiting cellular siroheme.« less

Authors:
 [1];  [1];  [1];  [2];  [3];  [4];  [1]
  1. Florida State Univ., Tallahassee, FL (United States)
  2. Florida State Univ., Tallahassee, FL (United States); National High Magnetic Field Lab. (MagLab), Tallahassee, FL (United States)
  3. National High Magnetic Field Lab. (MagLab), Tallahassee, FL (United States)
  4. Illinois Inst. of Technology, Chicago, IL (United States)
Publication Date:
Research Org.:
Argonne National Lab. (ANL), Argonne, IL (United States)
Sponsoring Org.:
National Science Foundation (NSF); USDOE Office of Science (SC), Basic Energy Sciences (BES) (SC-22). Scientific User Facilities Division
OSTI Identifier:
1418603
Grant/Contract Number:  
AC02-06CH11357
Resource Type:
Journal Article: Accepted Manuscript
Journal Name:
Journal of Biological Chemistry
Additional Journal Information:
Journal Volume: 290; Journal Issue: 31; Journal ID: ISSN 0021-9258
Publisher:
American Society for Biochemistry and Molecular Biology
Country of Publication:
United States
Language:
English
Subject:
59 BASIC BIOLOGICAL SCIENCES; 37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CHEMISTRY

Citation Formats

Askenasy, Isabel, Pennington, Joseph M., Tao, Yeqing, Marshall, Alan G., Young, Nicolas L., Shang, Weifeng, and Stroupe, M. Elizabeth. The N-terminal Domain of Escherichia coli Assimilatory NADPH-Sulfite Reductase Hemoprotein Is an Oligomerization Domain That Mediates Holoenzyme Assembly. United States: N. p., 2015. Web. doi:10.1074/jbc.M115.662379.
Askenasy, Isabel, Pennington, Joseph M., Tao, Yeqing, Marshall, Alan G., Young, Nicolas L., Shang, Weifeng, & Stroupe, M. Elizabeth. The N-terminal Domain of Escherichia coli Assimilatory NADPH-Sulfite Reductase Hemoprotein Is an Oligomerization Domain That Mediates Holoenzyme Assembly. United States. doi:10.1074/jbc.M115.662379.
Askenasy, Isabel, Pennington, Joseph M., Tao, Yeqing, Marshall, Alan G., Young, Nicolas L., Shang, Weifeng, and Stroupe, M. Elizabeth. Fri . "The N-terminal Domain of Escherichia coli Assimilatory NADPH-Sulfite Reductase Hemoprotein Is an Oligomerization Domain That Mediates Holoenzyme Assembly". United States. doi:10.1074/jbc.M115.662379. https://www.osti.gov/servlets/purl/1418603.
@article{osti_1418603,
title = {The N-terminal Domain of Escherichia coli Assimilatory NADPH-Sulfite Reductase Hemoprotein Is an Oligomerization Domain That Mediates Holoenzyme Assembly},
author = {Askenasy, Isabel and Pennington, Joseph M. and Tao, Yeqing and Marshall, Alan G. and Young, Nicolas L. and Shang, Weifeng and Stroupe, M. Elizabeth},
abstractNote = {Assimilatory NADPH-sulfite reductase (SiR) from Escherichia coli is a structurally complex oxidoreductase that catalyzes the six-electron reduction of sulfite to sulfide. Two subunits, one a flavin-binding flavoprotein (SiRFP, the α subunit) and the other an iron-containing hemoprotein (SiRHP, the β subunit), assemble to make a holoenzyme of about 800 kDa. How the two subunits assemble is not known. The iron-rich cofactors in SiRHP are unique because they are a covalent arrangement of a Fe4S4 cluster attached through a cysteine ligand to an iron-containing porphyrinoid called siroheme. The link between cofactor biogenesis and SiR stability is also ill-defined. By use of hydrogen/deuterium exchange and biochemical analysis, we show that the α8β4 SiR holoenzyme assembles through the N terminus of SiRHP and the NADPH binding domain of SiRFP. By use of small angle x-ray scattering, we explore the structure of the SiRHP N-terminal oligomerization domain. We additionally report a novel form of the hemoprotein that occurs in the absence of its cofactors. Apo-SiRHP forms a homotetramer, also dependent on its N terminus, that is unable to assemble with SiRFP. Finally, from these results, we propose that homotetramerization of apo-SiRHP serves as a quality control mechanism to prevent formation of inactive holoenzyme in the case of limiting cellular siroheme.},
doi = {10.1074/jbc.M115.662379},
journal = {Journal of Biological Chemistry},
issn = {0021-9258},
number = 31,
volume = 290,
place = {United States},
year = {2015},
month = {7}
}

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Works referenced in this record:

Construction of Escherichia coli K-12 in-frame, single-gene knockout mutants the Keio collection
journal, February 2006

  • Baba, Tomoya; Ara, Takeshi; Hasegawa, Miki
  • Molecular Systems Biology, Vol. 2, Article No. 2006.0008
  • DOI: 10.1038/msb4100050