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Title: Spectroscopic, thermodynamic and computational evidence of the locations of the FADs in the nitrogen fixation-associated electron transfer flavoprotein

Abstract

Flavin-based electron bifurcation allows enzymes to redistribute energy among electrons by coupling endergonic and exergonic electron transfer reactions. Diverse bifurcating enzymes employ a two-flavin electron transfer flavoprotein (ETF) that accepts hydride from NADH at a flavin (the so-called bifurcating FAD, Bf-FAD). The Bf-FAD passes one electron exergonically to a second flavin thereby assuming a reactive semiquinone state able to reduce ferredoxin or flavodoxin semiquinone. The flavin that accepts one electron and passes it on via exergonic electron transfer is known as the electron transfer FAD (ET-FAD) and is believed to correspond to the single FAD present in canonical ETFs, in domain II. The Bf-FAD is believed to be the one that is unique to bifurcating ETFs, bound between domains I and III. This very reasonable model has yet to be challenged experimentally. Herein we used site-directed mutagenesis to disrupt FAD binding to the presumed Bf site between domains I and III, in the Bf-ETF from Rhodopseudomonas palustris (RpaETF). The resulting protein contained only 0.80 ± 0.05 FAD, plus 1.21 ± 0.04 bound AMP as in canonical ETFs. The flavin was not subject to reduction by NADH, confirming absence of Bf-FAD. The retained FAD displayed visible circular dichroism (CD) similar tomore » that of the ET-FAD of RpaETF. Likewise, the mutant underwent two sequential one-electron reductions forming and then consuming anionic semiquinone, reproducing the reactivity of the ET-FAD. These data confirm that the retained FAD in domain II corresponds the ET-FAD. Quantum chemical calculations of the absorbance and CD spectra of each of WT RpaETF's two flavins reproduced the observed differences between their CD and absorbance signatures. The calculations for the flavin bound in domain II agreed better with the spectra of the ET-flavin, and those calculated based on the flavin between domains I and III agreed better with spectra of the Bf-flavin. Thus calculations independently confirm the locations of each flavin. We conclude that the site in domain II harbours the ET-FAD whereas the mutated site between domains I and III is the Bf-FAD site, confirming the accepted model by two different tests.« less

Authors:
ORCiD logo [1]; ORCiD logo [1]; ORCiD logo [2]; ORCiD logo [2]; ORCiD logo [3]
  1. Dept. Chemistry, University of Kentucky, Lexington, USA
  2. Max Volmer Laboratorum für Biophysikalische Chemie, Technische Universität – Berlin, 10623 Berlin, Germany
  3. Dept. Chemistry, University of Kentucky, Lexington, USA, Max Volmer Laboratorum für Biophysikalische Chemie
Publication Date:
Research Org.:
Montana State Univ., Bozeman, MT (United States)
Sponsoring Org.:
USDOE Office of Science (SC)
OSTI Identifier:
1532431
Alternate Identifier(s):
OSTI ID: 1611835
Grant/Contract Number:  
SC0012518
Resource Type:
Published Article
Journal Name:
Chemical Science
Additional Journal Information:
Journal Name: Chemical Science Journal Volume: 10 Journal Issue: 33; Journal ID: ISSN 2041-6520
Publisher:
Royal Society of Chemistry
Country of Publication:
United Kingdom
Language:
English
Subject:
37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CHEMISTRY; Chemistry

Citation Formats

Mohamed-Raseek, Nishya, Duan, H. Diessel, Hildebrandt, Peter, Mroginski, Maria Andrea, and Miller, Anne-Frances. Spectroscopic, thermodynamic and computational evidence of the locations of the FADs in the nitrogen fixation-associated electron transfer flavoprotein. United Kingdom: N. p., 2019. Web. doi:10.1039/C9SC00942F.
Mohamed-Raseek, Nishya, Duan, H. Diessel, Hildebrandt, Peter, Mroginski, Maria Andrea, & Miller, Anne-Frances. Spectroscopic, thermodynamic and computational evidence of the locations of the FADs in the nitrogen fixation-associated electron transfer flavoprotein. United Kingdom. doi:10.1039/C9SC00942F.
Mohamed-Raseek, Nishya, Duan, H. Diessel, Hildebrandt, Peter, Mroginski, Maria Andrea, and Miller, Anne-Frances. Wed . "Spectroscopic, thermodynamic and computational evidence of the locations of the FADs in the nitrogen fixation-associated electron transfer flavoprotein". United Kingdom. doi:10.1039/C9SC00942F.
@article{osti_1532431,
title = {Spectroscopic, thermodynamic and computational evidence of the locations of the FADs in the nitrogen fixation-associated electron transfer flavoprotein},
author = {Mohamed-Raseek, Nishya and Duan, H. Diessel and Hildebrandt, Peter and Mroginski, Maria Andrea and Miller, Anne-Frances},
abstractNote = {Flavin-based electron bifurcation allows enzymes to redistribute energy among electrons by coupling endergonic and exergonic electron transfer reactions. Diverse bifurcating enzymes employ a two-flavin electron transfer flavoprotein (ETF) that accepts hydride from NADH at a flavin (the so-called bifurcating FAD, Bf-FAD). The Bf-FAD passes one electron exergonically to a second flavin thereby assuming a reactive semiquinone state able to reduce ferredoxin or flavodoxin semiquinone. The flavin that accepts one electron and passes it on via exergonic electron transfer is known as the electron transfer FAD (ET-FAD) and is believed to correspond to the single FAD present in canonical ETFs, in domain II. The Bf-FAD is believed to be the one that is unique to bifurcating ETFs, bound between domains I and III. This very reasonable model has yet to be challenged experimentally. Herein we used site-directed mutagenesis to disrupt FAD binding to the presumed Bf site between domains I and III, in the Bf-ETF from Rhodopseudomonas palustris (RpaETF). The resulting protein contained only 0.80 ± 0.05 FAD, plus 1.21 ± 0.04 bound AMP as in canonical ETFs. The flavin was not subject to reduction by NADH, confirming absence of Bf-FAD. The retained FAD displayed visible circular dichroism (CD) similar to that of the ET-FAD of RpaETF. Likewise, the mutant underwent two sequential one-electron reductions forming and then consuming anionic semiquinone, reproducing the reactivity of the ET-FAD. These data confirm that the retained FAD in domain II corresponds the ET-FAD. Quantum chemical calculations of the absorbance and CD spectra of each of WT RpaETF's two flavins reproduced the observed differences between their CD and absorbance signatures. The calculations for the flavin bound in domain II agreed better with the spectra of the ET-flavin, and those calculated based on the flavin between domains I and III agreed better with spectra of the Bf-flavin. Thus calculations independently confirm the locations of each flavin. We conclude that the site in domain II harbours the ET-FAD whereas the mutated site between domains I and III is the Bf-FAD site, confirming the accepted model by two different tests.},
doi = {10.1039/C9SC00942F},
journal = {Chemical Science},
number = 33,
volume = 10,
place = {United Kingdom},
year = {2019},
month = {8}
}

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DOI: 10.1039/C9SC00942F

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Works referenced in this record:

Inter-Excited-State Phosphorescence in the Four-Component Relativistic Kohn–Sham Approximation: A Case Study on Lumiflavin
journal, November 2015

  • Falklöf, Olle; Durbeej, Bo; Norman, Patrick
  • The Journal of Physical Chemistry A, Vol. 119, Issue 49
  • DOI: 10.1021/acs.jpca.5b08908

The electron transfer flavoprotein: Ubiquinone oxidoreductases
journal, December 2010

  • Watmough, Nicholas J.; Frerman, Frank E.
  • Biochimica et Biophysica Acta (BBA) - Bioenergetics, Vol. 1797, Issue 12
  • DOI: 10.1016/j.bbabio.2010.10.007

Interaction between NADH and electron-transferring flavoprotein from Megasphaera elsdenii
journal, March 2013

  • Sato, K.; Nishina, Y.; Shiga, K.
  • Journal of Biochemistry, Vol. 153, Issue 6
  • DOI: 10.1093/jb/mvt026

Oxidation of the FAD cofactor to the 8-formyl-derivative in human electron-transferring flavoprotein
journal, January 2018

  • Augustin, Peter; Toplak, Marina; Fuchs, Katharina
  • Journal of Biological Chemistry, Vol. 293, Issue 8
  • DOI: 10.1074/jbc.RA117.000846

Unusual redox properties of electron-transfer flavoprotein from Methylophilus methylotrophus
journal, October 1989

  • Byron, Colleen M.; Stankovich, Marian T.; Husain, Mazhar
  • Biochemistry, Vol. 28, Issue 21
  • DOI: 10.1021/bi00447a047

Mechanistic insights into energy conservation by flavin-based electron bifurcation
journal, April 2017

  • Lubner, Carolyn E.; Jennings, David P.; Mulder, David W.
  • Nature Chemical Biology, Vol. 13, Issue 6
  • DOI: 10.1038/nchembio.2348

Density-functional exchange-energy approximation with correct asymptotic behavior
journal, September 1988


Single Hydrogen Bond Donation from Flavin N 5 to Proximal Asparagine Ensures FAD Reduction in DNA Photolyase
journal, March 2016

  • Wijaya, I. M. Mahaputra; Domratcheva, Tatiana; Iwata, Tatsuya
  • Journal of the American Chemical Society, Vol. 138, Issue 13
  • DOI: 10.1021/jacs.5b10533

Hybrid Models for Combined Quantum Mechanical and Molecular Mechanical Approaches
journal, January 1996

  • Bakowies, Dirk; Thiel, Walter
  • The Journal of Physical Chemistry, Vol. 100, Issue 25
  • DOI: 10.1021/jp9536514

Interaction of two arginine residues in lactate oxidase with the enzyme flavin: Conversion of FMN to 8-formyl-FMN
journal, November 2000

  • Yorita, K.; Matsuoka, T.; Misaki, H.
  • Proceedings of the National Academy of Sciences, Vol. 97, Issue 24
  • DOI: 10.1073/pnas.250472297

The SWISS-MODEL workspace: a web-based environment for protein structure homology modelling
journal, November 2005


Effects of protonation and deprotonation on the excitation energies of lumiflavin
journal, October 2008


Circular dichroism calculation for natural products
journal, April 2013


Dye chemistry with time-dependent density functional theory
journal, January 2014

  • Laurent, Adèle D.; Adamo, Carlo; Jacquemin, Denis
  • Phys. Chem. Chem. Phys., Vol. 16, Issue 28
  • DOI: 10.1039/C3CP55336A

Effect of a Charge-Transfer Interaction on the Catalytic Activity of Acyl-CoA Dehydrogenase:  A Theoretical Study of the Role of Oxidized Flavin
journal, November 2003

  • Dmitrenko, Olga; Thorpe, Colin; Bach, Robert D.
  • The Journal of Physical Chemistry B, Vol. 107, Issue 47
  • DOI: 10.1021/jp0348631

Self‐Consistent Molecular‐Orbital Methods. IX. An Extended Gaussian‐Type Basis for Molecular‐Orbital Studies of Organic Molecules
journal, January 1971

  • Ditchfield, R.; Hehre, W. J.; Pople, J. A.
  • The Journal of Chemical Physics, Vol. 54, Issue 2
  • DOI: 10.1063/1.1674902

An efficient implementation of time-dependent density-functional theory for the calculation of excitation energies of large molecules
journal, November 1998

  • Stratmann, R. Eric; Scuseria, Gustavo E.; Frisch, Michael J.
  • The Journal of Chemical Physics, Vol. 109, Issue 19
  • DOI: 10.1063/1.477483

Treatment of electronic excitations within the adiabatic approximation of time dependent density functional theory
journal, July 1996


Reduction of ferredoxin or oxygen by flavin-based electron bifurcation in Megasphaera elsdenii
journal, May 2015

  • Chowdhury, Nilanjan P.; Kahnt, Jörg; Buckel, Wolfgang
  • The FEBS Journal, Vol. 282, Issue 16
  • DOI: 10.1111/febs.13308

QM/MM Methods for Biomolecular Systems
journal, January 2009

  • Senn, Hans Martin; Thiel, Walter
  • Angewandte Chemie International Edition, Vol. 48, Issue 7
  • DOI: 10.1002/anie.200802019

A new form of mammalian electron-transferring flavoprotein
journal, February 1992


Interpretation of protein quantitation using the Bradford assay: Comparison with two calculation models
journal, March 2013


Molecular basis of the flavin-based electron-bifurcating caffeyl-CoA reductase reaction
journal, February 2018

  • Demmer, Julius K.; Bertsch, Johannes; Öppinger, Christian
  • FEBS Letters, Vol. 592, Issue 3
  • DOI: 10.1002/1873-3468.12971

Self‐consistent molecular orbital methods. XXIII. A polarization‐type basis set for second‐row elements
journal, October 1982

  • Francl, Michelle M.; Pietro, William J.; Hehre, Warren J.
  • The Journal of Chemical Physics, Vol. 77, Issue 7, p. 3654-3665
  • DOI: 10.1063/1.444267

Systematic Investigation of Modern Quantum Chemical Methods to Predict Electronic Circular Dichroism Spectra
journal, April 2003

  • Diedrich, Christian; Grimme, Stefan
  • The Journal of Physical Chemistry A, Vol. 107, Issue 14
  • DOI: 10.1021/jp0275802

Intrinsic Property of Flavin Mononucleotide Controls its Optical Spectra in Three Redox States
journal, September 2011


Defining Electron Bifurcation in the Electron-Transferring Flavoprotein Family
journal, August 2017

  • Garcia Costas, Amaya M.; Poudel, Saroj; Miller, Anne-Frances
  • Journal of Bacteriology, Vol. 199, Issue 21
  • DOI: 10.1128/JB.00440-17

UCSF Chimera?A visualization system for exploratory research and analysis
journal, January 2004

  • Pettersen, Eric F.; Goddard, Thomas D.; Huang, Conrad C.
  • Journal of Computational Chemistry, Vol. 25, Issue 13
  • DOI: 10.1002/jcc.20084

CHARMM36m: an improved force field for folded and intrinsically disordered proteins
journal, November 2016

  • Huang, Jing; Rauscher, Sarah; Nawrocki, Grzegorz
  • Nature Methods, Vol. 14, Issue 1
  • DOI: 10.1038/nmeth.4067

Quantum Mechanics/Molecular Mechanics Calculation of the Raman Spectra of the Phycocyanobilin Chromophore in α-C-Phycocyanin
journal, September 2007


The catalytic mechanism of electron-bifurcating electron transfer flavoproteins (ETFs) involves an intermediary complex with NAD +
journal, December 2018

  • Schut, Gerrit J.; Mohamed-Raseek, Nishya; Tokmina-Lukaszewska, Monika
  • Journal of Biological Chemistry, Vol. 294, Issue 9
  • DOI: 10.1074/jbc.RA118.005653

Long-range corrected hybrid density functionals with damped atom–atom dispersion corrections
journal, January 2008

  • Chai, Jeng-Da; Head-Gordon, Martin
  • Physical Chemistry Chemical Physics, Vol. 10, Issue 44
  • DOI: 10.1039/b810189b

Essential Genome of the Metabolically Versatile Alphaproteobacterium Rhodopseudomonas palustris
journal, December 2015

  • Pechter, Kieran B.; Gallagher, Larry; Pyles, Harley
  • Journal of Bacteriology, Vol. 198, Issue 5
  • DOI: 10.1128/JB.00771-15

Density‐functional thermochemistry. III. The role of exact exchange
journal, April 1993

  • Becke, Axel D.
  • The Journal of Chemical Physics, Vol. 98, Issue 7, p. 5648-5652
  • DOI: 10.1063/1.464913

Single-Reference ab Initio Methods for the Calculation of Excited States of Large Molecules
journal, November 2005

  • Dreuw, Andreas; Head-Gordon, Martin
  • Chemical Reviews, Vol. 105, Issue 11
  • DOI: 10.1021/cr0505627

Density functional theory calculation of electronic circular dichroism using London orbitals
journal, April 2004


TDDFT-calculations of Vis/NIR absorbing compounds
journal, January 2010


The Electron Bifurcating FixABCX Protein Complex from Azotobacter vinelandii : Generation of Low-Potential Reducing Equivalents for Nitrogenase Catalysis
journal, August 2017


Development of the Colle-Salvetti correlation-energy formula into a functional of the electron density
journal, January 1988


Distinct properties underlie flavin-based electron bifurcation in a novel electron transfer flavoprotein FixAB from Rhodopseudomonas palustris
journal, February 2018

  • Duan, H. Diessel; Lubner, Carolyn E.; Tokmina-Lukaszewska, Monika
  • Journal of Biological Chemistry, Vol. 293, Issue 13
  • DOI: 10.1074/jbc.RA117.000707

Acyl-CoA dehydrogenases. A mechanistic overview
journal, February 2004


Assessment of Franck–Condon Methods for Computing Vibrationally Broadened UV–vis Absorption Spectra of Flavin Derivatives: Riboflavin, Roseoflavin, and 5-Thioflavin
journal, November 2014

  • Karasulu, Bora; Götze, Jan Philipp; Thiel, Walter
  • Journal of Chemical Theory and Computation, Vol. 10, Issue 12
  • DOI: 10.1021/ct500830a

Zeolite Structure and Reactivity by Combined Quantum-Chemical−Classical Calculations
journal, July 1999

  • de Vries, Alex H.; Sherwood, Paul; Collins, Simon J.
  • The Journal of Physical Chemistry B, Vol. 103, Issue 29
  • DOI: 10.1021/jp9913012

Studies on the Mechanism of Electron Bifurcation Catalyzed by Electron Transferring Flavoprotein (Etf) and Butyryl-CoA Dehydrogenase (Bcd) of Acidaminococcus fermentans
journal, December 2013

  • Chowdhury, Nilanjan Pal; Mowafy, Amr M.; Demmer, Julius K.
  • Journal of Biological Chemistry, Vol. 289, Issue 8
  • DOI: 10.1074/jbc.M113.521013

(TD-)DFT Calculation of Vibrational and Vibronic Spectra of Riboflavin in Solution
journal, August 2010

  • Klaumünzer, Bastian; Kröner, Dominik; Saalfrank, Peter
  • The Journal of Physical Chemistry B, Vol. 114, Issue 33
  • DOI: 10.1021/jp100642c

QUASI: A general purpose implementation of the QM/MM approach and its application to problems in catalysis
journal, August 2003

  • Sherwood, Paul; de Vries, Alex H.; Guest, Martyn F.
  • Journal of Molecular Structure: THEOCHEM, Vol. 632, Issue 1-3
  • DOI: 10.1016/S0166-1280(03)00285-9

Spectroscopic Evidence for Changes in the Redox State of the Nitrogenase P-Cluster during Turnover
journal, May 1999

  • Chan, Jeannine M.; Christiansen, Jason; Dean, Dennis R.
  • Biochemistry, Vol. 38, Issue 18
  • DOI: 10.1021/bi982866b

The Current State of Ab Initio Calculations of Optical Rotation and Electronic Circular Dichroism Spectra
journal, December 2007

  • Crawford, T. Daniel; Tam, Mary C.; Abrams, Micah L.
  • The Journal of Physical Chemistry A, Vol. 111, Issue 48
  • DOI: 10.1021/jp075046u

Extensive TD-DFT Benchmark: Singlet-Excited States of Organic Molecules
journal, August 2009

  • Jacquemin, Denis; Wathelet, Valérie; Perpète, Eric A.
  • Journal of Chemical Theory and Computation, Vol. 5, Issue 9
  • DOI: 10.1021/ct900298e

SAC-CI theoretical study on the excited states of lumiflavin: Structure, excitation spectrum, and solvation effect
journal, June 2007

  • Hasegawa, Jun-ya; Bureekaew, Sareeya; Nakatsuji, Hiroshi
  • Journal of Photochemistry and Photobiology A: Chemistry, Vol. 189, Issue 2-3
  • DOI: 10.1016/j.jphotochem.2007.01.033

Investigation of the UV/Visible Absorption Spectra of Merocyanine Dyes Using Time-Dependent Density Functional Theory
journal, December 2006

  • Guillaume, Maxime; Champagne, Benoît; Zutterman, Freddy
  • The Journal of Physical Chemistry A, Vol. 110, Issue 48
  • DOI: 10.1021/jp064059p

SWISS-MODEL: homology modelling of protein structures and complexes
journal, May 2018

  • Waterhouse, Andrew; Bertoni, Martino; Bienert, Stefan
  • Nucleic Acids Research, Vol. 46, Issue W1
  • DOI: 10.1093/nar/gky427

The Binding of Adenine Nucleotides to Apo-Electron-Transferring Flavoprotein1
journal, December 1992


Analytical energy gradient of the ground, excited, ionized and electron-attached states calculated by the SAC/SAC-CI method
journal, November 1997


Electron-Transferring Flavoprotein Has an AMP-Binding Site in Addition to the FAD-Binding Site1
journal, August 1993


The semiquinone swing in the bifurcating electron transferring flavoprotein/butyryl-CoA dehydrogenase complex from Clostridium difficile
journal, November 2017

  • Demmer, Julius K.; Pal Chowdhury, Nilanjan; Selmer, Thorsten
  • Nature Communications, Vol. 8, Issue 1
  • DOI: 10.1038/s41467-017-01746-3

Three-dimensional structure of human electron transfer flavoprotein to 2.1-A resolution
journal, December 1996

  • Roberts, D. L.; Frerman, F. E.; Kim, J. -J. P.
  • Proceedings of the National Academy of Sciences, Vol. 93, Issue 25
  • DOI: 10.1073/pnas.93.25.14355

Electronic spectroscopy of lumiflavin in superfluid helium nanodroplets
journal, August 2013