Efficient and reversible electron bifurcation with either normal or inverted potentials at the bifurcating cofactor

Yuly, Jonathon L.; Zhang, Peng; Ru, Xuyan; Terai, Kiriko; Singh, Niven; Beratan, David N.

doi:10.1016/j.chempr.2021.03.016

Cited by 6 publications

(9 citation statements)

References 70 publications

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“…Conversely, the second electron must exhibit low potential and readily picked up by O 2 . However, new computational models predict that sites with normally distributed (uncrossed) potentials are EB-competent as well . Considering that proton-coupled electron transfer must occur during both formate oxidation and O 2 reduction, we posit that inverted potentials likely dominate at the Wco site during catalysis.…”

Section: Resultsmentioning

confidence: 90%

“…Other factors are also known to diminish stoichiometry of coupling in bifurcating [FeFe] hydrogenases . Finally, despite the current focus on reversible EB, irreversibility has entered the discussion . O 2 reduction enabled during FBEB (Bcd-EtfAB) , and MBEB (this work) underscores that irreversible EB deserves consideration (also see the Impact section).…”

Section: Resultsmentioning

confidence: 99%

“…However, new computational models predict that sites with normally distributed (uncrossed) potentials are EB-competent as well. 136 Considering that proton-coupled electron transfer 137 must occur during both formate oxidation and O 2 reduction, we posit that inverted potentials likely dominate at the Wco site during catalysis. Consistent with this assessment, an extremely weak W(V) EPR signature is observed with aerobically or anaerobically formate-reduced DvH-FDH2 (Figure 4).…”

Section: W(vi) E W(v) +mentioning

confidence: 98%

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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: 90%

Section: Resultsmentioning

confidence: 99%

Section: W(vi) E W(v) +mentioning

confidence: 98%

See 1 more Smart Citation

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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“…In fact, for several flavoproteins of the dataset only E m was available. Indeed, E 1 and E 2 are hard to measure, for example, because the flavoprotein reacts as a two-electron redox species, or the cofactor is characterized by crossed redox potential. − …”

Section: Resultsmentioning

confidence: 99%

Machine Learning for Efficient Prediction of Protein Redox Potential: The Flavoproteins Case

Galuzzi

Mirarchi

Viganò

et al. 2022

J. Chem. Inf. Model.

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Determining the redox potentials of protein cofactors and how they are influenced by their molecular neighborhoods is essential for basic research and many biotechnological applications, from biosensors and biocatalysis to bioremediation and bioelectronics. The laborious determination of redox potential with current experimental technologies pushes forward the need for computational approaches that can reliably predict it. Although current computational approaches based on quantum and molecular mechanics are accurate, their large computational costs hinder their usage. In this work, we explored the possibility of using more efficient QSPR models based on machine learning (ML) for the prediction of protein redox potential, as an alternative to classical approaches. As a proof of concept, we focused on flavoproteins, one of the most important families of enzymes directly involved in redox processes. To train and test different ML models, we retrieved a dataset of flavoproteins with a known midpoint redox potential (E m) and 3D structure. The features of interest, accounting for both short- and long-range effects of the protein matrix on the flavin cofactor, have been automatically extracted from each protein PDB file. Our best ML model (XGB) has a performance error below 1 kcal/mol (∼36 mV), comparing favorably to more sophisticated computational approaches. We also provided indications on the features that mostly affect the E m value, and when possible, we rationalized them on the basis of previous studies.

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“…This is accompanied by a conformational change so that the FMN cannot transfer electrons to the C1 cluster (Figure 11), as demonstrated by the NiFe-Bfu structure (Feng et al, 2022). We propose that the FMN in the NAD(H) bound form is uncrossed, meaning that the HQ/SQ couple is of lower potential than the SQ/OX couple (Yuly et al, 2021). The lower potential HQ/SQ couple can reduce the mid-potential B1 cluster while the high potential SQ/OX couple cannot, resulting in a stable SQ (state 9).…”

Section: A Unifying Electron Bifurcating Mechanism For Bfu Enzymesmentioning

confidence: 95%

An Abundant and Diverse New Family of Electron Bifurcating Enzymes With a Non-canonical Catalytic Mechanism

et al. 2022

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Microorganisms utilize electron bifurcating enzymes in metabolic pathways to carry out thermodynamically unfavorable reactions. Bifurcating FeFe-hydrogenases (HydABC) reversibly oxidize NADH (E′∼−280 mV, under physiological conditions) and reduce protons to H2 gas (E°′−414 mV) by coupling this endergonic reaction to the exergonic reduction of protons by reduced ferredoxin (Fd) (E′∼−500 mV). We show here that HydABC homologs are surprisingly ubiquitous in the microbial world and are represented by 57 phylogenetically distinct clades but only about half are FeFe-hydrogenases. The others have replaced the hydrogenase domain with another oxidoreductase domain or they contain additional subunits, both of which enable various third reactions to be reversibly coupled to NAD+ and Fd reduction. We hypothesize that all of these enzymes carry out electron bifurcation and that their third substrates can include hydrogen peroxide, pyruvate, carbon monoxide, aldehydes, aryl-CoA thioesters, NADP+, cofactor F420, formate, and quinones, as well as many yet to be discovered. Some of the enzymes are proposed to be integral membrane-bound proton-translocating complexes. These different functionalities are associated with phylogenetically distinct clades and in many cases with specific microbial phyla. We propose that this new and abundant class of electron bifurcating enzyme be referred to as the Bfu family whose defining feature is a conserved bifurcating BfuBC core. This core contains FMN and six iron sulfur clusters and it interacts directly with ferredoxin (Fd) and NAD(H). Electrons to or from the third substrate are fed into the BfuBC core via BfuA. The other three known families of electron bifurcating enzyme (abbreviated as Nfn, EtfAB, and HdrA) contain a special FAD that bifurcates electrons to high and low potential pathways. The Bfu family are proposed to use a different electron bifurcation mechanism that involves a combination of FMN and three adjacent iron sulfur clusters, including a novel [2Fe-2S] cluster with pentacoordinate and partial non-Cys coordination. The absolute conservation of the redox cofactors of BfuBC in all members of the Bfu enzyme family indicate they have the same non-canonical mechanism to bifurcate electrons. A hypothetical catalytic mechanism is proposed as a basis for future spectroscopic analyses of Bfu family members.

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Efficient and reversible electron bifurcation with either normal or inverted potentials at the bifurcating cofactor

Cited by 6 publications

References 70 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

Machine Learning for Efficient Prediction of Protein Redox Potential: The Flavoproteins Case

An Abundant and Diverse New Family of Electron Bifurcating Enzymes With a Non-canonical Catalytic Mechanism

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Efficient and reversible electron bifurcation with either normal or inverted potentials at the bifurcating cofactor

Cited by 6 publications

References 70 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

Machine Learning for Efficient Prediction of Protein Redox Potential: The Flavoproteins Case

An Abundant and Diverse New Family of Electron Bifurcating Enzymes With a Non-canonical Catalytic Mechanism

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