Noncovalent interactions that tune the reactivities of the flavins in bifurcating electron transferring flavoprotein

González-Viegas, María; Kar, Rajiv K.; Miller, Anne‐Frances; Mroginski, Maria‐Andrea

doi:10.1016/j.jbc.2023.104762

Cited by 4 publications

(9 citation statements)

References 73 publications

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“…The absence of ferredoxin reduction with the R140Q and R140M variants clearly demonstrates that this residue is indeed critical in maintaining the environment around the BF-FAD necessary for bifurcation, likely due to the insufficiently low potential of BF-FAD •– such that it cannot reduce ferredoxin. This is also suggested by a computational study indicating that the conserved arginine that hydrogen bonds to the N5 of the BF-FAD might be important to maintain the hydroquinone state of BF-FAD . At the same time, the present work indicates that reduction of the other redox-active centers in EtfABCX is also affected upon reduction of the complex by NADH, even though all can be fully reduced by the chemical reductant dithionite.…”

Section: Discussionsupporting

confidence: 79%

Characterization of the Membrane-Associated Electron-Bifurcating Flavoenzyme EtfABCX from the Hyperthermophilic Bacterium Thermotoga maritima

Ge,

Schut,

Tran

et al. 2023

Biochemistry

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Electron bifurcation is an energy-conservation mechanism in which a single enzyme couples an exergonic reaction with an endergonic one. Heterotetrameric EtfABCX drives the reduction of low-potential ferredoxin ( E °′ ∼ −450 mV) by oxidation of the midpotential NADH ( E °′ = −320 mV) by simultaneously coupling the reaction to reduction of the high-potential menaquinone ( E °′ = −74 mV). Electron bifurcation occurs at the NADH-oxidizing bifurcating-flavin adenine dinucleotide (BF-FAD) in EtfA, which has extremely crossed half-potentials and passes the first, high-potential electron to an electron-transferring FAD and via two iron–sulfur clusters eventually to menaquinone. The low-potential electron on the BF-FAD semiquinone simultaneously reduces ferredoxin. We have expressed the genes encoding Thermotoga maritima EtfABCX in E. coli and purified the EtfABCX holoenzyme and the EtfAB subcomplex. The bifurcation activity of EtfABCX was demonstrated by using electron paramagnetic resonance (EPR) to follow accumulation of reduced ferredoxin. To elucidate structural factors that impart the bifurcating ability, EPR and NADH titrations monitored by visible spectroscopy and dye-linked enzyme assays have been employed to characterize four conserved residues, R38, P239, and V242 in EtfA and R140 in EtfB, in the immediate vicinity of the BF-FAD. The R38, P239, and V242 variants showed diminished but still significant bifurcation activity. Despite still being partially reduced by NADH, the R140 variant had no bifurcation activity, and electron transfer to its two [4Fe-4S] clusters was prevented. The role of R140 is discussed in terms of the bifurcation mechanism in EtfABCX and in the other three families of bifurcating enzymes.

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Section: Discussionsupporting

confidence: 79%

Characterization of the Membrane-Associated Electron-Bifurcating Flavoenzyme EtfABCX from the Hyperthermophilic Bacterium Thermotoga maritima

Ge,

Schut,

Tran

et al. 2023

Biochemistry

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“…37 Meanwhile, the H290 near O2 is positioned to favour such a charge migration by donating a H-bond to the flavin O2. 6,28 Therefore, we hypothesized that if a methide intermediate is essential, then H290F Y279I should not undergo modification. This was confirmed by comparison of flavin modification yields in WT and H290F Y279I .…”

Section: Resultsmentioning

confidence: 99%

“…This is predicted to stabilize ASQ specifically, because the AHQ state tends to concentrate excess electron density near N5 instead, in consequence of N5's protonation. 28 Thus, we propose that His290 (H290) of Afe ETF is crucial for its stable ET-FAD ASQ state (ET ASQ ) and 1e − reactivity.…”

Section: Introductionmentioning

confidence: 99%

“…2 for flavin position numbering, amino acids' numbering is from Afe ETF, and the specified residues all derive from the A subunit). 16,23–28 However, these interactions do not suffice to explain the stability of ET-FAD's ASQ, as ASQ accumulates even when these residues are replaced. 14,26 Moreover, the positive charge of the Arg would stabilize ET-FAD's AHQ state as well as its ASQ, and the H-bond from Ser/Thr is weak based on computations.…”

Section: Introductionmentioning

confidence: 99%

“…14,26 Moreover, the positive charge of the Arg would stabilize ET-FAD's AHQ state as well as its ASQ, and the H-bond from Ser/Thr is weak based on computations. 28…”

Section: Introductionmentioning

confidence: 99%

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A single hydrogen bond that tunes flavin redox reactivity and activates it for modification

Das,

Miller

2024

Chem. Sci.

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Structures and Electron Transport Paths in the Four Families of Flavin-Based Electron Bifurcation Enzymes

Feng,

Schut,

Adams

et al. 2024

Subcellular Biochemistry

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Noncovalent interactions that tune the reactivities of the flavins in bifurcating electron transferring flavoprotein

Cited by 4 publications

References 73 publications

Characterization of the Membrane-Associated Electron-Bifurcating Flavoenzyme EtfABCX from the Hyperthermophilic Bacterium Thermotoga maritima

Characterization of the Membrane-Associated Electron-Bifurcating Flavoenzyme EtfABCX from the Hyperthermophilic Bacterium Thermotoga maritima

A single hydrogen bond that tunes flavin redox reactivity and activates it for modification

Structures and Electron Transport Paths in the Four Families of Flavin-Based Electron Bifurcation Enzymes

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