Structure-based electron-confurcation mechanism of the Ldh-EtfAB complex
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Evaluation Summary:
This paper describes a new structure for a complex between a bifurcating electron transfer flavoprotein (ETF) and its client dehydrogenase. Because electrons are being supplied by the dehydrogenase, the ETF executes confurcation in contrast to all of those elucidated so far, which function in the opposite direction to effect bifurcation. As electron-confurcation and electron-bifurcation have emerged as important paradigms of cellular bioenergetics, the data reported herein pave the way for future exploration of similar electron transfer systems and lay the ground for understanding their structural basis. The work will be of relevance to all who are interested in the mechanisms of enzymes.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #1 and Reviewer #3 agreed to share their name with the authors.)
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Abstract
Lactate oxidation with NAD + as electron acceptor is a highly endergonic reaction. Some anaerobic bacteria overcome the energetic hurdle by flavin-based electron bifurcation/confurcation (FBEB/FBEC) using a lactate dehydrogenase (Ldh) in concert with the electron-transferring proteins EtfA and EtfB. The electron cryo-microscopically characterized (Ldh-EtfAB) 2 complex of Acetobacterium woodii at 2.43 Å resolution consists of a mobile EtfAB shuttle domain located between the rigid central Ldh and the peripheral EtfAB base units. The FADs of Ldh and the EtfAB shuttle domain contact each other thereby forming the D (dehydrogenation-connected) state. The intermediary Glu37 and Glu139 may harmonize the redox potentials between the FADs and the pyruvate/lactate pair crucial for FBEC. By integrating Alphafold2 calculations a plausible novel B (bifurcation-connected) state was obtained allowing electron transfer between the EtfAB base and shuttle FADs. Kinetic analysis of enzyme variants suggests a correlation between NAD + binding site and D-to-B-state transition implicating a 75° rotation of the EtfAB shuttle domain. The FBEC inactivity when truncating the ferredoxin domain of EtfA substantiates its role as redox relay. Lactate oxidation in Ldh is assisted by the catalytic base His423 and a metal center. On this basis, a comprehensive catalytic mechanism of the FBEC process was proposed.
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Evaluation Summary:
This paper describes a new structure for a complex between a bifurcating electron transfer flavoprotein (ETF) and its client dehydrogenase. Because electrons are being supplied by the dehydrogenase, the ETF executes confurcation in contrast to all of those elucidated so far, which function in the opposite direction to effect bifurcation. As electron-confurcation and electron-bifurcation have emerged as important paradigms of cellular bioenergetics, the data reported herein pave the way for future exploration of similar electron transfer systems and lay the ground for understanding their structural basis. The work will be of relevance to all who are interested in the mechanisms of enzymes.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private …
Evaluation Summary:
This paper describes a new structure for a complex between a bifurcating electron transfer flavoprotein (ETF) and its client dehydrogenase. Because electrons are being supplied by the dehydrogenase, the ETF executes confurcation in contrast to all of those elucidated so far, which function in the opposite direction to effect bifurcation. As electron-confurcation and electron-bifurcation have emerged as important paradigms of cellular bioenergetics, the data reported herein pave the way for future exploration of similar electron transfer systems and lay the ground for understanding their structural basis. The work will be of relevance to all who are interested in the mechanisms of enzymes.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #1 and Reviewer #3 agreed to share their name with the authors.)
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Reviewer #1 (Public Review):
In this manuscript, Müller and Ermler groups report an investigation of structures and functions of the electron-confurcation complex of Ldh-EtfAB by cryo-EM. Two conformations of the complex are elucidated in this report. One is the conformation related to the D (dehydrogenase conducting) state; this structure has been analyzed directly from the cryo-EM data. Another conformation is the B (bifurcation conducting) state which was derived by Alphafold2 calculations. Functions of residues proposed to be related to electron transfers in this complex were investigated using site-directly mutagenesis or peptide truncation. As electron-confurcation and electron-bifurcation have emerged as important paradigms of cellular bioenergetics, the data reported herein pave the way for future exploration of similar electron …
Reviewer #1 (Public Review):
In this manuscript, Müller and Ermler groups report an investigation of structures and functions of the electron-confurcation complex of Ldh-EtfAB by cryo-EM. Two conformations of the complex are elucidated in this report. One is the conformation related to the D (dehydrogenase conducting) state; this structure has been analyzed directly from the cryo-EM data. Another conformation is the B (bifurcation conducting) state which was derived by Alphafold2 calculations. Functions of residues proposed to be related to electron transfers in this complex were investigated using site-directly mutagenesis or peptide truncation. As electron-confurcation and electron-bifurcation have emerged as important paradigms of cellular bioenergetics, the data reported herein pave the way for future exploration of similar electron transfer systems and lay the ground for understanding structural biology related to them.
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Reviewer #2 (Public Review):
This nice description of a new structure ties observations to a reasonable mechanism. The authors point out the significance of the fact that this complex operates in the direction of confurcation rather than bifurcation, and relates it to reduction midpoint potentials. The methods are appropriate including a combination of mutagenesis, catalytic assays and cryoEM.
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Reviewer #3 (Public Review):
The NAD+-dependent lactate oxidation is thermodynamically not very favorable as the redox potentials of the NADH/NAD+ and lactate/pyruvate couples are quite similar. Therefore, there is no strong thermodynamic drive in either direction. Anaerobic fermentation solves this use through electron-bifurcation. Lactate dehydrogenase forms a "three-flavin" complex with an electron transferring flavoprotein. In the first reaction step, lactate reduces the flavin of the dehydrogenase. An electron is then transferred to the so-called shuttling flavin of the electron transferring flavoprotein. In the meantime, a high/medium potential ferredoxin donates an electron to the NAD-reducing flavin of the electron transferring flavoprotein, providing the energetic drive for the reaction. After two cycles, the NAD-reducing …
Reviewer #3 (Public Review):
The NAD+-dependent lactate oxidation is thermodynamically not very favorable as the redox potentials of the NADH/NAD+ and lactate/pyruvate couples are quite similar. Therefore, there is no strong thermodynamic drive in either direction. Anaerobic fermentation solves this use through electron-bifurcation. Lactate dehydrogenase forms a "three-flavin" complex with an electron transferring flavoprotein. In the first reaction step, lactate reduces the flavin of the dehydrogenase. An electron is then transferred to the so-called shuttling flavin of the electron transferring flavoprotein. In the meantime, a high/medium potential ferredoxin donates an electron to the NAD-reducing flavin of the electron transferring flavoprotein, providing the energetic drive for the reaction. After two cycles, the NAD-reducing flavin becomes fully reduced and thereby able to transfer a hydride anion to the NAD+ acceptor. This is a complicated process that requires finely tuned redox potentials, strategies to prevent unwanted side and backward reactions, and a mobile electron shuttle that exchanges electrons from one site to the other.
Experimentally, the manuscript reports on a major result as the investigated enzymes are oxygen-labile and all the experiments had to be done anaerobically. Combined with mutagenesis, the three-dimensional structure reveals several interesting features such as the presence of an iron-sulfur cluster that mediates flavin reduction the ferredoxin. Moreover, the structure leads to predictions about the conformational changes that underpin electron-shuttling during the catalytic reaction. In general, this is excellent structural work that adds considerably to our understanding of electron bifurcation, a hot topic in current enzymology.
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