The role of thermodynamic features on the functional activity of electron bifurcating enzymes

Wise, Courtney E.; Ledinina, Anastasia E.; Yuly, Jonathon L.; Artz, Jacob H.; Lubner, Carolyn E.

doi:10.1016/j.bbabio.2021.148377

Cited by 11 publications

(5 citation statements)

References 80 publications

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“…Some have used the term cooperative bidirectional one-by-one electron transfer to distinguish it from noncooperative counterparts, as well as unidirectional events . EB was originally proposed in 1976 to rationalize mitochondrial quinone-based (QB) energy coupling. , The present-day literature is largely focused on flavin-based (FB) EB, − which is believed to have been discovered in 2008. However, one of us (C.S.R.)…”

Section: Resultsmentioning

confidence: 99%

How a Formate Dehydrogenase Responds to Oxygen: Unexpected O₂ Insensitivity of an Enzyme Harboring Tungstopterin, Selenocysteine, and [4Fe–4S] Clusters

et al. 2022

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The reversible two-electron interconversion of formate and CO2 is catalyzed by both nonmetallo- and metallo-formate dehydrogenases (FDHs). The latter group comprises molybdenum- or tungsten-containing enzymes with the metal coordinated by two equivalents of a pyranopterin cofactor, a cysteinyl or selenocysteinyl (Sec) ligand supplied by the polypeptide, and a catalytically essential terminal sulfido ligand. In addition, these biocatalysts incorporate one or more [4Fe–4S] clusters for facilitating long-distance electron transfer. However, an interesting dichotomy arises when attempting to understand how the metallo-FDHs react with O2. Whereas existing scholarship portrays these enzymes as being unable to perform in air due to extreme O2 lability of their metal centers, studies dating as far back as the 1930s emphasize that some of these systems exhibit formate oxidase (FOX) activity, coupling formate oxidation to O2 reduction. Therefore, to reconcile these conflicting views, we explored context-dependent functional linkages between metallo-FDHs and their cognate electron acceptors within the same organism vis-à-vis catalysis under atmospheric O2. Here, we report the discovery and characterization of an O2-insensitive FDH2 from the sulfate-reducing bacterium Desulfovibrio vulgaris Hildenborough (DvH) that ligates tungsten, Sec, and four [4Fe–4S] clusters. By advancing a robust expression platform for its recombinant production, we eliminate both the requirement of nitrate or azide during purification and reductive activation with thiols and/or formate prior to catalysis. Because the distinctive spectral signatures of formate-reduced DvH-FDH2 remain invariant under anaerobic and aerobic conditions, we benchmarked the enzyme activity in air, identifying CO2 as the catalytic product. Full reaction progress curve analysis discloses a high catalytic efficiency when probed with a high-potential artificial electron acceptor. Furthermore, we show that DvH-FDH2 enables near-stoichiometric hydrogen peroxide production without superoxide release to achieve O2 insensitivity. Notably, simultaneous electron transfer to cytochrome c and O2 reveals that metal-based electron bifurcation is operational in this system. Taken together, our work proves the co-occurrence of redox bifurcated FDH and FOX activities within a metalloenzyme scaffold. These findings set the stage for uncovering previously unknown O2-insensitive flavin-based electron bifurcation mechanisms, as well as for developing authentic formate/air biofuel cells, engineering O2-stable FDHs and biohybrid metallocatalysts, and discerning formate bioenergetics of gut microbiota.

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Section: Resultsmentioning

confidence: 99%

How a Formate Dehydrogenase Responds to Oxygen: Unexpected O₂ Insensitivity of an Enzyme Harboring Tungstopterin, Selenocysteine, and [4Fe–4S] Clusters

et al. 2022

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“…Electron bifurcation ( Wise et al, 2021 ) represents an alternative energy coupling mechanism to the well-known chemiosmotic coupling principle ( Rich, 2003 ). Electron bifurcation drives thermodynamically unfavorable (endergonic) redox reactions by coupling them to energetically favorable (exergonic) redox reactions directly within the same enzyme.…”

Section: Introductionmentioning

confidence: 99%

Structural insight on the mechanism of an electron-bifurcating [FeFe] hydrogenase

Furlan

Chongdar

Gupta

et al. 2022

eLife

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Electron-bifurcation is a fundamental energy conservation mechanism in nature in which two electrons from an intermediate potential electron donor are split so that one is sent along a high potential pathway to a high potential acceptor and the other is sent along a low potential pathway to a low potential acceptor. This process allows endergonic reactions to be driven by exergonic ones and is an alternative, less recognised, mechanism of energy coupling to the well-known chemiosmotic principle. The electron-bifurcating [FeFe] hydrogenase from Thermotoga maritima (HydABC) requires both NADH and ferredoxin to reduce protons generating hydrogen. The mechanism of electron-bifurcation in HydABC remains enigmatic in spite of intense research efforts over the last few years. Structural information may provide the basis for a better understanding of spectroscopic and functional information. Here, we present a 2.3 Å electron cryo-microscopy structure of HydABC. The structure shows a heterododecamer composed of two independent 'halves' each made of two strongly interacting HydABC heterotrimers connected via a [4Fe-4S] cluster. A central electron transfer pathway connects the active sites for NADH oxidation and for proton reduction. We identified two conformations of a flexible iron-sulfur cluster domain: a 'closed bridge' and an 'open bridge' conformation, where a Zn2+ site may act as a 'hinge' allowing domain movement. Based on these structural revelations, we propose a possible mechanism of electron-bifurcation in HydABC where the flavin mononucleotide serves a dual role as both the electron bifurcation center and as the NAD+ reduction/NADH oxidation site.

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“…Despite flattening of the NfnSL energy landscape for the initial electron transfer for the first bifurcated electron, the steep thermodynamically uphill nature of the overall high-potential branch in NfnSL is preserved, as the terminal electron acceptor of this pathway, NAD + , possesses a formal reduction potential of −320 mV. Further, the overall magnitude of the uphill electron transfer events across the high-potential branch (from bifurcating cofactor SQ/HQ to high-potential branch substrate) is comparable between our newly established NfnSL landscape and the quinone-based cytochrome bc 1 complex, at ∼+300 mV ( 8 ). Short-circuit reactions have been proposed to be influenced by the semiquinone stability constant, K S ( Eq.…”

Section: Resultsmentioning

confidence: 64%

“…Enzymes drive energetically challenging chemical reactions with high efficiency and fidelity by employing innovative solutions for overcoming thermodynamic and kinetic barriers. This is particularly true for electron bifurcating enzymes, which separate a pair of electrons at a single cofactor and direct each along spatially and thermodynamically distinct pathways that conclude with delivery of the individual electrons to different products ( 1 – 8 ). Bifurcating systems utilize the free energy generated from an initial exergonic oxidation-reduction reaction along a high-potential catalytic branch to drive a coupled endergonic one across a low-potential pathway ( 9 , 10 ).…”

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confidence: 99%

An uncharacteristically low-potential flavin governs the energy landscape of electron bifurcation

Wise

Ledinina

Mulder

et al. 2022

Proc. Natl. Acad. Sci. U.S.A.

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Significance Nature has long been an inspiration for materials design, as it exemplifies exquisite control of both matter and energy. Electron bifurcation, a mechanism employed in biological systems to drive thermodynamically unfavorable and energetically challenging chemical reactions, is one such example. A key feature of bifurcating enzymes is the ability of a single redox cofactor to distribute a pair of electrons across two spatially separated electron transfer pathways. Here, we report on the empirical determination of both the one-electron potential and two-electron potential of the bifurcating flavin cofactor in the NADH-dependent ferredoxin-NADP + oxidoreductase I (NfnSL) enzyme. Insights arising from the defined energy landscape of this bifurcation site may underlie the design of synthetic catalysts capable of generating high-energy intermediates.

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The role of thermodynamic features on the functional activity of electron bifurcating enzymes

Cited by 11 publications

References 80 publications

How a Formate Dehydrogenase Responds to Oxygen: Unexpected O₂ Insensitivity of an Enzyme Harboring Tungstopterin, Selenocysteine, and [4Fe–4S] Clusters

How a Formate Dehydrogenase Responds to Oxygen: Unexpected O₂ Insensitivity of an Enzyme Harboring Tungstopterin, Selenocysteine, and [4Fe–4S] Clusters

Structural insight on the mechanism of an electron-bifurcating [FeFe] hydrogenase

An uncharacteristically low-potential flavin governs the energy landscape of electron bifurcation

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The role of thermodynamic features on the functional activity of electron bifurcating enzymes

Cited by 11 publications

References 80 publications

How a Formate Dehydrogenase Responds to Oxygen: Unexpected O2 Insensitivity of an Enzyme Harboring Tungstopterin, Selenocysteine, and [4Fe–4S] Clusters

How a Formate Dehydrogenase Responds to Oxygen: Unexpected O2 Insensitivity of an Enzyme Harboring Tungstopterin, Selenocysteine, and [4Fe–4S] Clusters

Structural insight on the mechanism of an electron-bifurcating [FeFe] hydrogenase

An uncharacteristically low-potential flavin governs the energy landscape of electron bifurcation

Contact Info

Product

Resources

About

How a Formate Dehydrogenase Responds to Oxygen: Unexpected O₂ Insensitivity of an Enzyme Harboring Tungstopterin, Selenocysteine, and [4Fe–4S] Clusters

How a Formate Dehydrogenase Responds to Oxygen: Unexpected O₂ Insensitivity of an Enzyme Harboring Tungstopterin, Selenocysteine, and [4Fe–4S] Clusters