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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]
+ Show Author Affiliations
  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.
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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. https://doi.org/10.1039/C9SC00942F
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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. https://doi.org/10.1039/C9SC00942F.
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@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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