Spectroscopic, thermodynamic and computational evidence of the locations of the FADs in the nitrogen fixation-associated electron transfer flavoprotein
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Mohamed-Raseek, Nishya
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Univ Kentucky, Dept Chem, 505 Rose St, Lexington, KY 40506 USAUniv Kentucky, Dept Chem, 505 Rose St, Lexington, KY 40506 USA
Mohamed-Raseek, Nishya
[1
]
Duan, H. Diessel
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Univ Kentucky, Dept Chem, 505 Rose St, Lexington, KY 40506 USAUniv Kentucky, Dept Chem, 505 Rose St, Lexington, KY 40506 USA
Duan, H. Diessel
[1
]
Hildebrandt, Peter
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Tech Univ Berlin, Max Volmer Lab Biophys Chem, Sekr PC 14,135 Str 17 Juni, D-10623 Berlin, GermanyUniv Kentucky, Dept Chem, 505 Rose St, Lexington, KY 40506 USA
Hildebrandt, Peter
[2
]
Mroginski, Maria Andrea
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Tech Univ Berlin, Max Volmer Lab Biophys Chem, Sekr PC 14,135 Str 17 Juni, D-10623 Berlin, GermanyUniv Kentucky, Dept Chem, 505 Rose St, Lexington, KY 40506 USA
Mroginski, Maria Andrea
[2
]
Miller, Anne-Frances
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Univ Kentucky, Dept Chem, 505 Rose St, Lexington, KY 40506 USA
Tech Univ Berlin, Max Volmer Lab Biophys Chem, Sekr PC 14,135 Str 17 Juni, D-10623 Berlin, GermanyUniv Kentucky, Dept Chem, 505 Rose St, Lexington, KY 40506 USA
Miller, Anne-Frances
[1
,2
]
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[1] Univ Kentucky, Dept Chem, 505 Rose St, Lexington, KY 40506 USA
[2] Tech Univ Berlin, Max Volmer Lab Biophys Chem, Sekr PC 14,135 Str 17 Juni, D-10623 Berlin, Germany
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.
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Royal Inst Technol, Sch Biotechnol, S-10691 Stockholm, SwedenBeijing Normal Univ, Coll Chem, Beijing 100875, Peoples R China
Ai, Yue-jie
Tian, Guangjun
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Royal Inst Technol, Sch Biotechnol, S-10691 Stockholm, SwedenBeijing Normal Univ, Coll Chem, Beijing 100875, Peoples R China
Tian, Guangjun
Liao, Rong-zhen
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Stockholm Univ, Dept Organ Chem, S-10691 Stockholm, SwedenBeijing Normal Univ, Coll Chem, Beijing 100875, Peoples R China
Liao, Rong-zhen
Zhang, Qiong
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Royal Inst Technol, Sch Biotechnol, S-10691 Stockholm, SwedenBeijing Normal Univ, Coll Chem, Beijing 100875, Peoples R China
Zhang, Qiong
Fang, Wei-hai
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Beijing Normal Univ, Coll Chem, Beijing 100875, Peoples R ChinaBeijing Normal Univ, Coll Chem, Beijing 100875, Peoples R China
Fang, Wei-hai
Luo, Yi
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Royal Inst Technol, Sch Biotechnol, S-10691 Stockholm, Sweden
Univ Sci & Technol China, Heifei Natl Lab Phys Sci Microscale, Hefei 230026, Anhui, Peoples R ChinaBeijing Normal Univ, Coll Chem, Beijing 100875, Peoples R China
机构:
Royal Inst Technol, Sch Biotechnol, S-10691 Stockholm, SwedenBeijing Normal Univ, Coll Chem, Beijing 100875, Peoples R China
Ai, Yue-jie
Tian, Guangjun
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机构:
Royal Inst Technol, Sch Biotechnol, S-10691 Stockholm, SwedenBeijing Normal Univ, Coll Chem, Beijing 100875, Peoples R China
Tian, Guangjun
Liao, Rong-zhen
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Stockholm Univ, Dept Organ Chem, S-10691 Stockholm, SwedenBeijing Normal Univ, Coll Chem, Beijing 100875, Peoples R China
Liao, Rong-zhen
Zhang, Qiong
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Royal Inst Technol, Sch Biotechnol, S-10691 Stockholm, SwedenBeijing Normal Univ, Coll Chem, Beijing 100875, Peoples R China
Zhang, Qiong
Fang, Wei-hai
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Beijing Normal Univ, Coll Chem, Beijing 100875, Peoples R ChinaBeijing Normal Univ, Coll Chem, Beijing 100875, Peoples R China
Fang, Wei-hai
Luo, Yi
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机构:
Royal Inst Technol, Sch Biotechnol, S-10691 Stockholm, Sweden
Univ Sci & Technol China, Heifei Natl Lab Phys Sci Microscale, Hefei 230026, Anhui, Peoples R ChinaBeijing Normal Univ, Coll Chem, Beijing 100875, Peoples R China