Search Menu
DOE PAGES title logo U.S. Department of Energy
Office of Scientific and Technical Information
Search Scholarly Publications

Title: Neutron scattering maps the higher-order assembly of NADPH-dependent assimilatory sulfite reductase

Abstract

Precursor molecules for biomass incorporation must be imported into cells and made available to the molecular machines that build the cell. Sulfur-containing macromolecules require that sulfur be in its S2- oxidation state before assimilation into amino acids, cofactors, and vitamins that are essential to organisms throughout the biosphere. In α-proteobacteria, NADPH-dependent assimilatory sulfite reductase (SiR) performs the final six-electron reduction of sulfur. SiR is a dodecameric oxidoreductase composed of an octameric flavoprotein reductase (SiRFP) and four hemoprotein metalloenzyme oxidases (SiRHPs). SiR performs the electron transfer reduction reaction to produce sulfide from sulfite through coordinated domain movements and subunit interactions without release of partially reduced intermediates. Efforts to understand the electron transfer mechanism responsible for SiR’s efficiency are confounded by structural heterogeneity arising from intrinsically disordered regions throughout its complex, including the flexible linker joining SiRFP’s flavin-binding domains. As a result, high-resolution structures of SiR dodecamer and its subcomplexes are unknown, leaving a gap in the fundamental understanding of how SiR performs this uniquely large-volume electron transfer reaction. In this work, we use deuterium labeling, in vitro reconstitution, analytical ultracentrifugation (AUC), small-angle neutron scattering (SANS), and neutron contrast variation (NCV) to observe the relative subunit positions within SiR’s higher-order assembly. AUCmore » and SANS reveal SiR to be a flexible dodecamer and confirm the mismatched SiRFP and SiRHP subunit stoichiometry. NCV shows that the complex is asymmetric, with SiRHP on the periphery of the complex and the centers of mass between SiRFP and SiRHP components over 100 Å apart. SiRFP undergoes compaction upon assembly into SiR’s dodecamer and SiRHP adopts multiple positions in the complex. The resulting map of SiR’s higher-order structure supports a cis/trans mechanism for electron transfer between domains of reductase subunits as well as between tightly bound or transiently interacting reductase and oxidase subunits.« less

Authors:
 [1];  [1]; ORCiD logo [2]; ORCiD logo [2];  [1]; ORCiD logo [2]; ORCiD logo [1]
+ Show Author Affiliations
  1. Florida State Univ., Tallahassee, FL (United States)
  2. Oak Ridge National Lab. (ORNL), Oak Ridge, TN (United States)
Publication Date:
Research Org.:
Oak Ridge National Lab. (ORNL), Oak Ridge, TN (United States)
Sponsoring Org.:
USDOE Office of Science (SC), Biological and Environmental Research (BER); National Science Foundation (NSF)
OSTI Identifier:
1869099
Grant/Contract Number:  
AC05-00OR22725; MCB1856502; CHE1904612
Resource Type:
Accepted Manuscript
Journal Name:
Biophysical Journal
Additional Journal Information:
Journal Volume: 121; Journal Issue: 10; Journal ID: ISSN 0006-3495
Publisher:
Elsevier
Country of Publication:
United States
Language:
English
Subject:
59 BASIC BIOLOGICAL SCIENCES

Citation Formats

Murray, Daniel T., Walia, Nidhi, Weiss, Kevin L., Stanley, Christopher B., Randolph, Peter S., Nagy, Gergely, and Stroupe, M. Elizabeth. Neutron scattering maps the higher-order assembly of NADPH-dependent assimilatory sulfite reductase. United States: N. p., 2022. Web. doi:10.1016/j.bpj.2022.04.021.
Copy to clipboard
Murray, Daniel T., Walia, Nidhi, Weiss, Kevin L., Stanley, Christopher B., Randolph, Peter S., Nagy, Gergely, & Stroupe, M. Elizabeth. Neutron scattering maps the higher-order assembly of NADPH-dependent assimilatory sulfite reductase. United States. https://doi.org/10.1016/j.bpj.2022.04.021
Copy to clipboard
Murray, Daniel T., Walia, Nidhi, Weiss, Kevin L., Stanley, Christopher B., Randolph, Peter S., Nagy, Gergely, and Stroupe, M. Elizabeth. Wed . "Neutron scattering maps the higher-order assembly of NADPH-dependent assimilatory sulfite reductase". United States. https://doi.org/10.1016/j.bpj.2022.04.021. https://www.osti.gov/servlets/purl/1869099.
Copy to clipboard
@article{osti_1869099,
title = {Neutron scattering maps the higher-order assembly of NADPH-dependent assimilatory sulfite reductase},
author = {Murray, Daniel T. and Walia, Nidhi and Weiss, Kevin L. and Stanley, Christopher B. and Randolph, Peter S. and Nagy, Gergely and Stroupe, M. Elizabeth},
abstractNote = {Precursor molecules for biomass incorporation must be imported into cells and made available to the molecular machines that build the cell. Sulfur-containing macromolecules require that sulfur be in its S2- oxidation state before assimilation into amino acids, cofactors, and vitamins that are essential to organisms throughout the biosphere. In α-proteobacteria, NADPH-dependent assimilatory sulfite reductase (SiR) performs the final six-electron reduction of sulfur. SiR is a dodecameric oxidoreductase composed of an octameric flavoprotein reductase (SiRFP) and four hemoprotein metalloenzyme oxidases (SiRHPs). SiR performs the electron transfer reduction reaction to produce sulfide from sulfite through coordinated domain movements and subunit interactions without release of partially reduced intermediates. Efforts to understand the electron transfer mechanism responsible for SiR’s efficiency are confounded by structural heterogeneity arising from intrinsically disordered regions throughout its complex, including the flexible linker joining SiRFP’s flavin-binding domains. As a result, high-resolution structures of SiR dodecamer and its subcomplexes are unknown, leaving a gap in the fundamental understanding of how SiR performs this uniquely large-volume electron transfer reaction. In this work, we use deuterium labeling, in vitro reconstitution, analytical ultracentrifugation (AUC), small-angle neutron scattering (SANS), and neutron contrast variation (NCV) to observe the relative subunit positions within SiR’s higher-order assembly. AUC and SANS reveal SiR to be a flexible dodecamer and confirm the mismatched SiRFP and SiRHP subunit stoichiometry. NCV shows that the complex is asymmetric, with SiRHP on the periphery of the complex and the centers of mass between SiRFP and SiRHP components over 100 Å apart. SiRFP undergoes compaction upon assembly into SiR’s dodecamer and SiRHP adopts multiple positions in the complex. The resulting map of SiR’s higher-order structure supports a cis/trans mechanism for electron transfer between domains of reductase subunits as well as between tightly bound or transiently interacting reductase and oxidase subunits.},
doi = {10.1016/j.bpj.2022.04.021},
journal = {Biophysical Journal},
number = 10,
volume = 121,
place = {United States},
year = {Wed Apr 20 00:00:00 EDT 2022},
month = {Wed Apr 20 00:00:00 EDT 2022}
}
Copy to clipboard
Journal Article:
Free Publicly Available Full Text
Publisher's Version of Record
https://doi.org/10.1016/j.bpj.2022.04.021
Copyright Statement
Other availability
Search WorldCat Search WorldCat to find libraries that may hold this journal
Save / Share:
Export Metadata
Save to My Library
You must Sign In or Create an Account in order to save documents to your library.

Works referenced in this record:

The full-length cytochrome P450 enzyme CYP102A1 dimerizes at its reductase domains and has flexible heme domains for efficient catalysis
journal, May 2018

  • Zhang, Haoming; Yokom, Adam L.; Cheng, Shen
  • Journal of Biological Chemistry, Vol. 293, Issue 20
  • DOI: 10.1074/jbc.RA117.000600

Mantid—Data analysis and visualization package for neutron scattering and μ SR experiments
journal, November 2014

  • Arnold, O.; Bilheux, J. C.; Borreguero, J. M.
  • Nuclear Instruments and Methods in Physics Research Section A: Accelerators, Spectrometers, Detectors and Associated Equipment, Vol. 764
  • DOI: 10.1016/j.nima.2014.07.029

Ab initio electron density determination directly from solution scattering data
journal, January 2018

Global Rigid Body Modeling of Macromolecular Complexes against Small-Angle Scattering Data
journal, August 2005

Analytical ultracentrifugation in structural biology
journal, November 2017

Absolute calibration of small-angle neutron scattering data
journal, February 1987

Determination of the regularization parameter in indirect-transform methods using perceptual criteria
journal, August 1992

Accuracy of molecular mass determination of proteins in solution by small-angle X-ray scattering
journal, February 2007

  • Mylonas, Efstratios; Svergun, Dmitri I.
  • Journal of Applied Crystallography, Vol. 40, Issue s1
  • DOI: 10.1107/S002188980700252X

BioXTAS RAW : improvements to a free open-source program for small-angle X-ray scattering data reduction and analysis
journal, September 2017

  • Hopkins, Jesse Bennett; Gillilan, Richard E.; Skou, Soren
  • Journal of Applied Crystallography, Vol. 50, Issue 5
  • DOI: 10.1107/S1600576717011438

A Simplifed Functional Version of the Escherichia coli Sulfite Reductase
journal, September 2000

  • Zeghouf, Mahel; Fontecave, Marc; Covès, Jacques
  • Journal of Biological Chemistry, Vol. 275, Issue 48
  • DOI: 10.1074/jbc.M005619200

Sedimentation velocity analysis of highly heterogeneous systems
journal, December 2004

  • Demeler, Borries; van Holde, Kensal E.
  • Analytical Biochemistry, Vol. 335, Issue 2, p. 279-288
  • DOI: 10.1016/j.ab.2004.08.039

Fatty acid synthase, a proficient multifunctional enzyme
journal, May 1989

NADPH–cytochrome P450 oxidoreductase: Prototypic member of the diflavin reductase family
journal, December 2012

  • Iyanagi, Takashi; Xia, Chuanwu; Kim, Jung-Ja P.
  • Archives of Biochemistry and Biophysics, Vol. 528, Issue 1
  • DOI: 10.1016/j.abb.2012.09.002

A designed heme-[4Fe-4S] metalloenzyme catalyzes sulfite reduction like the native enzyme
journal, September 2018

SASBDB: Towards an automatically curated and validated repository for biological scattering data
journal, October 2019

  • Kikhney, Alexey G.; Borges, Clemente R.; Molodenskiy, Dmitry S.
  • Protein Science, Vol. 29, Issue 1
  • DOI: 10.1002/pro.3731

The “bacterial heterodisulfide” DsrC is a key protein in dissimilatory sulfur metabolism
journal, July 2014

  • Venceslau, S. S.; Stockdreher, Y.; Dahl, C.
  • Biochimica et Biophysica Acta (BBA) - Bioenergetics, Vol. 1837, Issue 7
  • DOI: 10.1016/j.bbabio.2014.03.007

Reduced Nicotinamide Adenine Dinucleotide Phosphate-Sulfite Reductase of Enterobacteria
journal, March 1974

The N-terminal Domain of Escherichia coli Assimilatory NADPH-Sulfite Reductase Hemoprotein Is an Oligomerization Domain That Mediates Holoenzyme Assembly
journal, July 2015

  • Askenasy, Isabel; Pennington, Joseph M.; Tao, Yeqing
  • Journal of Biological Chemistry, Vol. 290, Issue 31
  • DOI: 10.1074/jbc.M115.662379

MULCh : modules for the analysis of small-angle neutron contrast variation data from biomolecular assemblies
journal, January 2008

  • Whitten, Andrew E.; Cai, Shuzhi; Trewhella, Jill
  • Journal of Applied Crystallography, Vol. 41, Issue 1
  • DOI: 10.1107/S0021889807055136

X-ray solution scattering (SAXS) combined with crystallography and computation: defining accurate macromolecular structures, conformations and assemblies in solution
journal, August 2007

  • Putnam, Christopher D.; Hammel, Michal; Hura, Greg L.
  • Quarterly Reviews of Biophysics, Vol. 40, Issue 3
  • DOI: 10.1017/S0033583507004635

Protein release and foaming in Escherichia coli cultures grown in minimal medium
journal, October 1996

  • Törnkvist, M.; Larsson, G.; Enfors, S. -O.
  • Bioprocess Engineering, Vol. 15, Issue 5
  • DOI: 10.1007/BF02391583

Structural Insights into Dissimilatory Sulfite Reductases: Structure of Desulforubidin from Desulfomicrobium Norvegicum
journal, January 2011

  • Oliveira, Tânia F.; Franklin, Edward; Afonso, José P.
  • Frontiers in Microbiology, Vol. 2
  • DOI: 10.3389/fmicb.2011.00071

Predictable Deuteration of Recombinant Proteins Expressed inEscherichia coli
journal, December 1998

  • Leiting, Barbara; Marsilio, Frank; O'Connell, John F.
  • Analytical Biochemistry, Vol. 265, Issue 2
  • DOI: 10.1006/abio.1998.2904

Architecture of the Nitric-oxide Synthase Holoenzyme Reveals Large Conformational Changes and a Calmodulin-driven Release of the FMN Domain
journal, June 2014

  • Yokom, Adam L.; Morishima, Yoshihiro; Lau, Miranda
  • Journal of Biological Chemistry, Vol. 289, Issue 24
  • DOI: 10.1074/jbc.M114.564005

Redox-Linked Domain Movements in the Catalytic Cycle of Cytochrome P450 Reductase
journal, September 2013

On the estimation of the radius of gyration of the subunits of macromolecular aggregates of biological origin in situ
journal, August 1981

The extended Q -range small-angle neutron scattering diffractometer at the SNS
journal, July 2010

Sulfite Reductase Structure at 1.6  : Evolution and Catalysis for Reduction of Inorganic Anions
journal, October 1995

Mutational Analysis of Sulfite Reductase Hemoprotein Reveals the Mechanism for Coordinated Electron and Proton Transfer
journal, November 2012

  • Smith, Kyle W.; Stroupe, M. Elizabeth
  • Biochemistry, Vol. 51, Issue 49
  • DOI: 10.1021/bi300947a

C9orf72 Poly(PR) Dipeptide Repeats Disturb Biomolecular Phase Separation and Disrupt Nucleolar Function
journal, May 2019

Reduced Nicotinamide Adenine Dinucleotide Phosphate-Sulfite Reductase of Enterobacteria
journal, January 1973

Orchestrated Domain Movement in Catalysis by Cytochrome P450 Reductase
journal, August 2017

Comparison of neutron and X-ray scattering of dilute myoglobin solutions
journal, April 1975

The suite of small-angle neutron scattering instruments at Oak Ridge National Laboratory
journal, February 2018

  • Heller, William T.; Cuneo, Matthew; Debeer-Schmitt, Lisa
  • Journal of Applied Crystallography, Vol. 51, Issue 2
  • DOI: 10.1107/S1600576718001231

Asymmetry in the 50S Ribosomal Subunit of Escherichia coli
journal, January 1974

  • Moore, P. B.; Engelman, D. M.; Schoenborn, B. P.
  • Proceedings of the National Academy of Sciences, Vol. 71, Issue 1
  • DOI: 10.1073/pnas.71.1.172

Enzymatic complexes across scales
journal, October 2018

  • Kastritis, Panagiotis L.; Gavin, Anne-Claude
  • Essays in Biochemistry, Vol. 62, Issue 4
  • DOI: 10.1042/EBC20180008

Revving up an artificial metalloenzyme
journal, September 2018

2017 publication guidelines for structural modelling of small-angle scattering data from biomolecules in solution: an update
journal, August 2017

  • Trewhella, Jill; Duff, Anthony P.; Durand, Dominique
  • Acta Crystallographica Section D Structural Biology, Vol. 73, Issue 9
  • DOI: 10.1107/S2059798317011597

Small-angle neutron scattering solution structures of NADPH-dependent sulfite reductase
journal, June 2021

  • Murray, Daniel T.; Weiss, Kevin L.; Stanley, Christopher B.
  • Journal of Structural Biology, Vol. 213, Issue 2
  • DOI: 10.1016/j.jsb.2021.107724

Reduced Nicotinamide Adenine Dinucleotide Phosphate-Sulfite Reductase of Enterobacteria
journal, March 1974

Troponin I Encompasses an Extended Troponin C in the Ca2+-Bound Complex: A Small-Angle X-ray and Neutron Scattering Study
journal, July 1994

  • Olah, Glenn A.; Rokop, Sue E.; Wang, C. -L. Albert
  • Biochemistry, Vol. 33, Issue 27
  • DOI: 10.1021/bi00193a009

SAXS experiments on absolute scale with Kratky systems using water as a secondary standard
journal, April 2000

  • Orthaber, Doris; Bergmann, Alexander; Glatter, Otto
  • Journal of Applied Crystallography, Vol. 33, Issue 2
  • DOI: 10.1107/S0021889899015216

UCSF ChimeraX: Meeting modern challenges in visualization and analysis: UCSF ChimeraX Visualization System
journal, September 2017

  • Goddard, Thomas D.; Huang, Conrad C.; Meng, Elaine C.
  • Protein Science, Vol. 27, Issue 1
  • DOI: 10.1002/pro.3235

Four crystal structures of the 60 kDa flavoprotein monomer of the sulfite reductase indicate a disordered flavodoxin-like module 1 1Edited by R. Huber
journal, May 2000

  • Gruez, Arnaud; Pignol, David; Zeghouf, Mahel
  • Journal of Molecular Biology, Vol. 299, Issue 1
  • DOI: 10.1006/jmbi.2000.3748

Structure and Function of an NADPH-Cytochrome P450 Oxidoreductase in an Open Conformation Capable of Reducing Cytochrome P450
journal, January 2009

  • Hamdane, Djemel; Xia, Chuanwu; Im, Sang-Choul
  • Journal of Biological Chemistry, Vol. 284, Issue 17
  • DOI: 10.1074/jbc.M807868200

The Structure of the KinA-Sda Complex Suggests an Allosteric Mechanism of Histidine Kinase Inhibition
journal, April 2007

  • Whitten, Andrew E.; Jacques, David A.; Hammouda, Boualem
  • Journal of Molecular Biology, Vol. 368, Issue 2
  • DOI: 10.1016/j.jmb.2007.01.064

Solution structure of the cytochrome P450 reductase–cytochrome c complex determined by neutron scattering
journal, April 2018

  • Freeman, Samuel L.; Martel, Anne; Devos, Juliette M.
  • Journal of Biological Chemistry, Vol. 293, Issue 14
  • DOI: 10.1074/jbc.RA118.001941

NADPH-dependent sulfite reductase flavoprotein adopts an extended conformation unique to this diflavin reductase
journal, February 2019

  • Tavolieri, Angela M.; Murray, Daniel T.; Askenasy, Isabel
  • Journal of Structural Biology, Vol. 205, Issue 2
  • DOI: 10.1016/j.jsb.2019.01.001

Reduced Nicotinamide Adenine Dinucleotide Phosphate-Sulfite Reductase of Enterobacteria
journal, April 1973

Three-dimensional structure of NADPH-cytochrome P450 reductase: Prototype for FMN- and FAD-containing enzymes
journal, August 1997

  • Wang, M.; Roberts, D. L.; Paschke, R.
  • Proceedings of the National Academy of Sciences, Vol. 94, Issue 16
  • DOI: 10.1073/pnas.94.16.8411

Conformational Changes of NADPH-Cytochrome P450 Oxidoreductase Are Essential for Catalysis and Cofactor Binding
journal, February 2011

  • Xia, Chuanwu; Hamdane, Djemel; Shen, Anna L.
  • Journal of Biological Chemistry, Vol. 286, Issue 18
  • DOI: 10.1074/jbc.M111.230532

A cross-domain charge interaction governs the activity of NO synthase
journal, March 2018

  • Haque, Mohammad Mahfuzul; Tejero, Jesús; Bayachou, Mekki
  • Journal of Biological Chemistry, Vol. 293, Issue 12
  • DOI: 10.1074/jbc.RA117.000635

Characterizing flexible and intrinsically unstructured biological macromolecules by SAS using the Porod-Debye law
journal, April 2011

  • Rambo, Robert P.; Tainer, John A.
  • Biopolymers, Vol. 95, Issue 8
  • DOI: 10.1002/bip.21638

Structure–Function Relationships in the Oligomeric NADPH-Dependent Assimilatory Sulfite Reductase
journal, May 2018

The Protein Data Bank
journal, January 2000

Molecular architecture of mammalian nitric oxide synthases
journal, August 2014

  • Campbell, M. G.; Smith, B. C.; Potter, C. S.
  • Proceedings of the National Academy of Sciences, Vol. 111, Issue 35
  • DOI: 10.1073/pnas.1413763111

Structural basis for human NADPH-cytochrome P450 oxidoreductase deficiency
journal, August 2011

  • Xia, C.; Panda, S. P.; Marohnic, C. C.
  • Proceedings of the National Academy of Sciences, Vol. 108, Issue 33
  • DOI: 10.1073/pnas.1106632108

ATSAS 3.0 : expanded functionality and new tools for small-angle scattering data analysis
journal, February 2021

  • Manalastas-Cantos, Karen; Konarev, Petr V.; Hajizadeh, Nelly R.
  • Journal of Applied Crystallography, Vol. 54, Issue 1
  • DOI: 10.1107/S1600576720013412
    Sort by:
    [ × clear filter / sort ]

    Similar Records in DOE PAGES and OSTI.GOV collections: