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

ABSTRACT: 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 ?-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-?-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, ?-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.

SUBMITTER: Toplak M

PROVIDER: S-EPMC6771786 | biostudies-literature | 2019 Sep

REPOSITORIES: biostudies-literature

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

Toplak Marina M Brunner Julia J Tabib Chaitanya R CR Macheroux Peter P

The FEBS journal 20190525 18

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-la ...[more]

PMID: 31081204

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Project description:Electron bifurcation is the coupling of exergonic and endergonic redox reactions to simultaneously generate (or utilize) low- and high-potential electrons. It is the third recognized form of energy conservation in biology and was recently described for select electron-transferring flavoproteins (Etfs). Etfs are flavin-containing heterodimers best known for donating electrons derived from fatty acid and amino acid oxidation to an electron transfer respiratory chain via Etf-quinone oxidoreductase. Canonical examples contain a flavin adenine dinucleotide (FAD) that is involved in electron transfer, as well as a non-redox-active AMP. However, Etfs demonstrated to bifurcate electrons contain a second FAD in place of the AMP. To expand our understanding of the functional variety and metabolic significance of Etfs and to identify amino acid sequence motifs that potentially enable electron bifurcation, we compiled 1,314 Etf protein sequences from genome sequence databases and subjected them to informatic and structural analyses. Etfs were identified in diverse archaea and bacteria, and they clustered into five distinct well-supported groups, based on their amino acid sequences. Gene neighborhood analyses indicated that these Etf group designations largely correspond to putative differences in functionality. Etfs with the demonstrated ability to bifurcate were found to form one group, suggesting that distinct conserved amino acid sequence motifs enable this capability. Indeed, structural modeling and sequence alignments revealed that identifying residues occur in the NADH- and FAD-binding regions of bifurcating Etfs. Collectively, a new classification scheme for Etf proteins that delineates putative bifurcating versus nonbifurcating members is presented and suggests that Etf-mediated bifurcation is associated with surprisingly diverse enzymes.IMPORTANCE Electron bifurcation has recently been recognized as an electron transfer mechanism used by microorganisms to maximize energy conservation. Bifurcating enzymes couple thermodynamically unfavorable reactions with thermodynamically favorable reactions in an overall spontaneous process. Here we show that the electron-transferring flavoprotein (Etf) enzyme family exhibits far greater diversity than previously recognized, and we provide a phylogenetic analysis that clearly delineates bifurcating versus nonbifurcating members of this family. Structural modeling of proteins within these groups reveals key differences between the bifurcating and nonbifurcating Etfs.

Dataset Information

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

Publications

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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