Enzyme-Mediated Conversion of Flavin Adenine Dinucleotide (FAD) to 8-Formyl FAD in Formate Oxidase Results in a Modified Cofactor with Enhanced Catalytic Properties

Robbins, John L.; Souffrant, Michael; Hamelberg, Donald; Gadda, Giovanni; Bommarius, Andreas S.

doi:10.1021/acs.biochem.7b00335

Cited by 28 publications

(47 citation statements)

References 29 publications

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“…We propose that the oxidation of the 8α-methyl group is initiated by proton abstraction, as proposed previously ( 16 – 18 ), leading to the formation of a negative charge at the N(1)–C(2)=O locus of the isoalloxazine ring. After addition of water, the reduced intermediate is oxidized to yield the 8α-hydroxymethylene intermediate, which is again subject to proton abstraction at the 8α-group with the resulting enol tautomerizing to the reduced 8f-FAD.…”

Section: Discussionsupporting

confidence: 65%

Oxidation of the FAD cofactor to the 8-formyl-derivative in human electron-transferring flavoprotein

Augustín

Toplak

Fuchs

et al. 2018

Journal of Biological Chemistry

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The heterodimeric human (h) electron-transferring flavoprotein (ETF) transfers electrons from at least 13 different flavin dehydrogenases to the mitochondrial respiratory chain through a non-covalently bound FAD cofactor. Here, we describe the discovery of an irreversible and pH-dependent oxidation of the 8α-methyl group to 8-formyl-FAD (8f-FAD), which represents a unique chemical modification of a flavin cofactor in the human flavoproteome. Furthermore, a set of hETF variants revealed that several conserved amino acid residues in the FAD-binding pocket of electron-transferring flavoproteins are required for the conversion to the formyl group. Two of the variants generated in our study, namely αR249C and αT266M, cause glutaric aciduria type II, a severe inherited disease. Both of the variants showed impaired formation of 8f-FAD shedding new light on the potential molecular cause of disease development. Interestingly, the conversion of FAD to 8f-FAD yields a very stable flavin semiquinone that exhibited slightly lower rates of electron transfer in an artificial assay system than hETF containing FAD. In contrast, the formation of 8f-FAD enhanced the affinity to human dimethylglycine dehydrogenase 5-fold, indicating that formation of 8f-FAD modulates the interaction of hETF with client enzymes in the mitochondrial matrix. Thus, we hypothesize that the FAD cofactor bound to hETF is subject to oxidation in the alkaline (pH 8) environment of the mitochondrial matrix, which may modulate electron transport between client dehydrogenases and the respiratory chain. This discovery challenges the current concepts of electron transfer processes in mitochondria.

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Section: Discussionsupporting

confidence: 65%

Oxidation of the FAD cofactor to the 8-formyl-derivative in human electron-transferring flavoprotein

Augustín

Toplak

Fuchs

et al. 2018

Journal of Biological Chemistry

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“…Amplitudes were fit to a single exponential decay in time. A shoulder around 520 nm persisted in the absorbance spectrum collected after heat-denaturation and the short-wavelength maximum was shifted to 360 nm vs 375 for authentic FAD consistent with possible modification of some of the flavin (48,114). (B) Near-UV CD spectra of the same sample as described in (A) with inset showing time course of tertiary structural change based on molar ellipticity at 280 nm.…”

Section: Data Availabilitymentioning

confidence: 77%

Spectroscopic evidence for direct flavin-flavin contact in a bifurcating electron transfer flavoprotein

Duan

Mohamed-Raseek

Miller

2020

Journal of Biological Chemistry

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A remarkable charge transfer (CT) band is described in the bifurcating electron transfer flavoprotein (Bf-ETF) from Rhodopseudomonas palustris (RpaETF). RpaETF contains two FADs that play contrasting roles in electron bifurcation. The Bf-FAD accepts electrons pairwise from NADH, directs one to a lower-E° carrier and the other to the higher-E° electron transfer FAD (ET-FAD). Previous work noted that the CT band at 726 nm formed when ET-FAD was reduced and Bf-FAD was oxidized, suggesting that both flavins participate. However, existing crystal structures place them too far apart to interact directly. We present biochemical experiments addressing this conundrum and elucidating the nature of this CT species. We observed that RpaETF missing either FAD lacked the 726 nm band. Site-directed mutagenesis near either FAD produced altered yields of the CT species, supporting involvement of both flavins. The residue substitutions did not alter the absorption maximum of the signal, ruling out contributions from residue orbitals. Instead, we propose that the residue identities modulate the population of a protein conformation that brings the ET-flavin and Bf-flavin into direct contact, explaining the 726 nm band based on a CT complex of reduced ET-FAD and oxidized Bf-FAD. This is corroborated by persistence of the 726 nm species during gentle protein denaturation and simple density functional theory calculations of flavin dimers. Although such a CT complex has been demonstrated for free flavins, this may be the first observation of such, to our knowledge, in an enzyme. Thus, Bf-ETFs may optimize electron transfer efficiency by enabling direct flavin-flavin contact.

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“…hETF is oxidized at the 8a-position to yield 8-formyl-FAD upon incubation under slightly alkaline conditions [17]. Interestingly, this reaction is promoted by bTyr16 in hETF [17,28,29] and thus we hypothesized that yETF may be resistant toward oxidation of the FAD cofactor. To test our hypothesis, we transferred purified yETF to 50 mM HEPES pH 8.5 containing 1 mM DTT and incubated the sample at 25°C for 24 h. After denaturation of the protein, the free cofactor was analyzed on HPLC-DAD, which indeed revealed no 8-formyl modification of the flavin cofactor.…”

Section: Comparison Of the Yeast Etf To The Human Homologmentioning

confidence: 97%

“…The only obvious difference found in the active site of the model is the change of Tyr16 in hETF to Phe19 in yETF in the b-subunit (Fig. Interestingly, this reaction is promoted by bTyr16 in hETF [17,28,29] and thus we hypothesized that yETF may be resistant toward oxidation of the FAD cofactor. Recently, we have shown that the FAD cofactor of with 50 mM HEPES pH 7 + 1 mM DTT (black line) and denatured using 2% SDS (red line) were recorded between 300 and 800 nm at 25°C.…”

Section: Comparison Of the Yeast Etf To The Human Homologmentioning

confidence: 98%

Closing the gap: yeast electron‐transferring flavoprotein links the oxidation of d‐lactate and d‐α‐hydroxyglutarate to energy production via the respiratory chain

et al. 2019

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Electron‐transferring flavoproteins (ETFs) have been found in all kingdoms of life, mostly assisting in shuttling electrons to the respiratory chain for ATP production. While the human (h) ETF has been studied in great detail, very little is known about the biochemical properties of the homologous protein in the model organism Saccharomyces cerevisiae (yETF). In view of the absence of client dehydrogenases, for example, the acyl‐CoA dehydrogenases involved in the β‐oxidation of fatty acids, d‐lactate dehydrogenase 2 (Dld2) appeared to be the only relevant enzyme that is serviced by yETF for electron transfer to the mitochondrial electron transport chain. However, this hypothesis was never tested experimentally. Here, we report the biochemical properties of yETF and Dld2 as well as the electron transfer reaction between the two proteins. Our study revealed that Dld2 oxidizes d‐α‐hydroxyglutarate more efficiently than d‐lactate exhibiting kcatapp/KMapp values of 1200 ± 300 m−1·s−1 and 11 ± 2 m−1·s−1, respectively. As expected, substrate‐reduced Dld2 very slowly reacted with oxygen or the artificial electron acceptor 2,6‐dichlorophenol indophenol. However, photoreduced Dld2 was rapidly reoxidized by oxygen, suggesting that the reaction products, that is, α‐ketoglutarate and pyruvate, ‘lock’ the reduced enzyme in an unreactive state. Interestingly, however, we could demonstrate that substrate‐reduced Dld2 rapidly transfers electrons to yETF. Therefore, we conclude that the formation of a product‐reduced Dld2 complex suppresses electron transfer to dioxygen but favors the rapid reduction in yETF, thus preventing the loss of electrons and the generation of reactive oxygen species.

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Enzyme-Mediated Conversion of Flavin Adenine Dinucleotide (FAD) to 8-Formyl FAD in Formate Oxidase Results in a Modified Cofactor with Enhanced Catalytic Properties

Cited by 28 publications

References 29 publications

Oxidation of the FAD cofactor to the 8-formyl-derivative in human electron-transferring flavoprotein

Oxidation of the FAD cofactor to the 8-formyl-derivative in human electron-transferring flavoprotein

Spectroscopic evidence for direct flavin-flavin contact in a bifurcating electron transfer flavoprotein

Closing the gap: yeast electron‐transferring flavoprotein links the oxidation of d‐lactate and d‐α‐hydroxyglutarate to energy production via the respiratory chain

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