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Entry
Database: EMDB / ID: EMD-15212
TitleCryo-EM structure of the electron bifurcating Fe-Fe hydrogenase HydABC complex from Thermoanaerobacter kivui in the reduced state
Map dataCryo-EM structure of the electron bifurcating Fe-Fe hydrogenase HydABC complex from Thermoanaerobacter kivui in the reduced state.
Sample
  • Complex: Electron bifurcating Fe-Fe hydrogenase HydABC complex from Thermoanaerobacter kivui
    • Protein or peptide: Electron bifurcating hydrogenase subunit HydA1
    • Protein or peptide: Electron bifurcating hydrogenase subunit HydB
    • Protein or peptide: Electron bifurcating hydrogenase subunit HydC
  • Ligand: IRON/SULFUR CLUSTERIron–sulfur cluster
  • Ligand: 2 IRON/2 SULFUR/5 CARBONYL/2 WATER INORGANIC CLUSTER
  • Ligand: FE2/S2 (INORGANIC) CLUSTER
  • Ligand: FLAVIN MONONUCLEOTIDE
  • Ligand: ZINC ION
  • Ligand: NADP NICOTINAMIDE-ADENINE-DINUCLEOTIDE PHOSPHATE
Function / homology
Function and homology information


hydrogen dehydrogenase (NADP+) / hydrogen dehydrogenase (NADP+) activity / cellular anatomical entity / ferredoxin hydrogenase activity / NADH dehydrogenase (ubiquinone) activity / ATP synthesis coupled electron transport / 2 iron, 2 sulfur cluster binding / FMN binding / 4 iron, 4 sulfur cluster binding / iron ion binding ...hydrogen dehydrogenase (NADP+) / hydrogen dehydrogenase (NADP+) activity / cellular anatomical entity / ferredoxin hydrogenase activity / NADH dehydrogenase (ubiquinone) activity / ATP synthesis coupled electron transport / 2 iron, 2 sulfur cluster binding / FMN binding / 4 iron, 4 sulfur cluster binding / iron ion binding / membrane / metal ion binding
Similarity search - Function
Iron hydrogenase, small subunit superfamily / Iron hydrogenase, subset / Iron hydrogenase, small subunit / Iron hydrogenase small subunit / Iron hydrogenase small subunit / Iron hydrogenase, large subunit, C-terminal / Iron hydrogenase / Iron only hydrogenase large subunit, C-terminal domain / 4Fe-4S binding domain / Soluble ligand binding domain ...Iron hydrogenase, small subunit superfamily / Iron hydrogenase, subset / Iron hydrogenase, small subunit / Iron hydrogenase small subunit / Iron hydrogenase small subunit / Iron hydrogenase, large subunit, C-terminal / Iron hydrogenase / Iron only hydrogenase large subunit, C-terminal domain / 4Fe-4S binding domain / Soluble ligand binding domain / SLBB domain / Respiratory-chain NADH dehydrogenase 75 Kd subunit signature 1. / NADH:ubiquinone oxidoreductase, 75kDa subunit, conserved site / NADH-ubiquinone oxidoreductase-G iron-sulfur binding region / NADH-ubiquinone oxidoreductase-G iron-sulfur binding region / Respiratory-chain NADH dehydrogenase 24 Kd subunit signature. / NuoE domain / NADH-quinone oxidoreductase subunit E-like / NADH-quinone oxidoreductase subunit E, N-terminal / NADH:ubiquinone oxidoreductase, subunit G, iron-sulphur binding / His(Cys)3-ligated-type [4Fe-4S] domain profile. / NADH:ubiquinone oxidoreductase, 51kDa subunit, conserved site / Respiratory-chain NADH dehydrogenase 51 Kd subunit signature 2. / NADH-ubiquinone oxidoreductase 51kDa subunit, FMN-binding domain / NADH-ubiquinone oxidoreductase 51kDa subunit, iron-sulphur binding domain / NADH-ubiquinone oxidoreductase 51kDa subunit, iron-sulphur binding domain superfamily / NADH-ubiquinone oxidoreductase 51kDa subunit, FMN-binding domain superfamily / Respiratory-chain NADH dehydrogenase 51 Kd subunit / NADH-ubiquinone oxidoreductase-F iron-sulfur binding region / NADH-ubiquinone oxidoreductase-F iron-sulfur binding region / 2Fe-2S ferredoxin-type iron-sulfur binding domain profile. / 2Fe-2S ferredoxin-type iron-sulfur binding domain / 2Fe-2S ferredoxin-like superfamily / 4Fe-4S ferredoxin, iron-sulphur binding, conserved site / 4Fe-4S ferredoxin-type iron-sulfur binding region signature. / 4Fe-4S ferredoxin-type iron-sulfur binding domain profile. / 4Fe-4S ferredoxin-type, iron-sulphur binding domain / Thioredoxin-like superfamily
Similarity search - Domain/homology
Electron bifurcating hydrogenase subunit HydA1 / Electron bifurcating hydrogenase subunit HydB / Electron bifurcating hydrogenase subunit HydC
Similarity search - Component
Biological speciesThermoanaerobacter kivui (bacteria)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.1 Å
AuthorsKumar A / Saura P / Gamiz-Hernandez AP / Kaila VRI / Mueller V / Schuller JM
Funding support Germany, European Union, Sweden, 6 items
OrganizationGrant numberCountry
German Research Foundation (DFG)SCHU 3364/1-1 Germany
European Research Council (ERC)715311 (VRIK)European Union
European Research Council (ERC)741791European Union
Knut and Alice Wallenberg FoundationVRIK Sweden
German Research Foundation (DFG)SFB1078 Germany
Swedish Research Council016-07213 Sweden
CitationJournal: J Am Chem Soc / Year: 2023
Title: Molecular Basis of the Electron Bifurcation Mechanism in the [FeFe]-Hydrogenase Complex HydABC.
Authors: Alexander Katsyv / Anuj Kumar / Patricia Saura / Maximilian C Pöverlein / Sven A Freibert / Sven T Stripp / Surbhi Jain / Ana P Gamiz-Hernandez / Ville R I Kaila / Volker Müller / Jan M Schuller /
Abstract: Electron bifurcation is a fundamental energy coupling mechanism widespread in microorganisms that thrive under anoxic conditions. These organisms employ hydrogen to reduce CO, but the molecular ...Electron bifurcation is a fundamental energy coupling mechanism widespread in microorganisms that thrive under anoxic conditions. These organisms employ hydrogen to reduce CO, but the molecular mechanisms have remained enigmatic. The key enzyme responsible for powering these thermodynamically challenging reactions is the electron-bifurcating [FeFe]-hydrogenase HydABC that reduces low-potential ferredoxins (Fd) by oxidizing hydrogen gas (H). By combining single-particle cryo-electron microscopy (cryoEM) under catalytic turnover conditions with site-directed mutagenesis experiments, functional studies, infrared spectroscopy, and molecular simulations, we show that HydABC from the acetogenic bacteria and employ a single flavin mononucleotide (FMN) cofactor to establish electron transfer pathways to the NAD(P) and Fd reduction sites by a mechanism that is fundamentally different from classical flavin-based electron bifurcation enzymes. By modulation of the NAD(P) binding affinity via reduction of a nearby iron-sulfur cluster, HydABC switches between the exergonic NAD(P) reduction and endergonic Fd reduction modes. Our combined findings suggest that the conformational dynamics establish a redox-driven kinetic gate that prevents the backflow of the electrons from the Fd reduction branch toward the FMN site, providing a basis for understanding general mechanistic principles of electron-bifurcating hydrogenases.
History
DepositionJun 19, 2022-
Header (metadata) releaseFeb 15, 2023-
Map releaseFeb 15, 2023-
UpdateMar 29, 2023-
Current statusMar 29, 2023Processing site: PDBe / Status: Released

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Structure visualization

Supplemental images

emd_15212.png

Downloads & links

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Map

FileDownload / File: emd_15212.map.gz / Format: CCP4 / Size: 83.7 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationCryo-EM structure of the electron bifurcating Fe-Fe hydrogenase HydABC complex from Thermoanaerobacter kivui in the reduced state.
Voxel sizeX=Y=Z: 0.87 Å
Density
Contour LevelBy AUTHOR: 0.0618
Minimum - Maximum-0.18352793 - 0.9486401
Average (Standard dev.)0.0013986517 (±0.018099848)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions280280280
Spacing280280280
CellA=B=C: 243.6 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: Half-map B

Fileemd_15212_half_map_1.map
AnnotationHalf-map B
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Half-map A

Fileemd_15212_half_map_2.map
AnnotationHalf-map A
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : Electron bifurcating Fe-Fe hydrogenase HydABC complex from Thermo...

EntireName: Electron bifurcating Fe-Fe hydrogenase HydABC complex from Thermoanaerobacter kivui
Components
  • Complex: Electron bifurcating Fe-Fe hydrogenase HydABC complex from Thermoanaerobacter kivui
    • Protein or peptide: Electron bifurcating hydrogenase subunit HydA1
    • Protein or peptide: Electron bifurcating hydrogenase subunit HydB
    • Protein or peptide: Electron bifurcating hydrogenase subunit HydC
  • Ligand: IRON/SULFUR CLUSTERIron–sulfur cluster
  • Ligand: 2 IRON/2 SULFUR/5 CARBONYL/2 WATER INORGANIC CLUSTER
  • Ligand: FE2/S2 (INORGANIC) CLUSTER
  • Ligand: FLAVIN MONONUCLEOTIDE
  • Ligand: ZINC ION
  • Ligand: NADP NICOTINAMIDE-ADENINE-DINUCLEOTIDE PHOSPHATE

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Supramolecule #1: Electron bifurcating Fe-Fe hydrogenase HydABC complex from Thermo...

SupramoleculeName: Electron bifurcating Fe-Fe hydrogenase HydABC complex from Thermoanaerobacter kivui
type: complex / ID: 1 / Chimera: Yes / Parent: 0 / Macromolecule list: #1-#3
Source (natural)Organism: Thermoanaerobacter kivui (bacteria)
Molecular weightTheoretical: 348 KDa

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Macromolecule #1: Electron bifurcating hydrogenase subunit HydA1

MacromoleculeName: Electron bifurcating hydrogenase subunit HydA1 / type: protein_or_peptide / ID: 1 / Number of copies: 2 / Enantiomer: LEVO / EC number: hydrogen dehydrogenase (NADP+)
Source (natural)Organism: Thermoanaerobacter kivui (bacteria)
Molecular weightTheoretical: 63.890727 KDa
SequenceString: MNMVMLTIDG KQVQVEKGTT IKKAAEKLGI EIPGLCDDND LKPFGACRLC VVEDARGNLV ASCHTPVREG MVVKTNSPKV LKARRVILE LLLSSHNADC FECDKNLHCK LQKYAYELNI RNIRFKGEKR NYEIKDNGPI YYDPNKCILC GKCVRICEEV Q HICAIDFA ...String:
MNMVMLTIDG KQVQVEKGTT IKKAAEKLGI EIPGLCDDND LKPFGACRLC VVEDARGNLV ASCHTPVREG MVVKTNSPKV LKARRVILE LLLSSHNADC FECDKNLHCK LQKYAYELNI RNIRFKGEKR NYEIKDNGPI YYDPNKCILC GKCVRICEEV Q HICAIDFA SRGFKAYIST PFEKPLLESD CIFCGQCVRV CPTGALAEKT DIERIYEAIS DPNKVVVVQV APAVRVALGE EF GLEPGEI VTGKMVAALK RLGFDKVFDT QFAADMTIVE ETAELVERLE KGENFPMFTS CCPSWILAVE KFYPELIPNI STA RSPQQI FGAIAKNYYA KKIGVARENM FVVSVMPCIG KKFEATRPEF NNDVDAVLTT RELARMIKES GIDFIKLEEE NFDS PLGES TGAAAIFGVT GGVMEAALRT AYSIMTGEEL EGDKIEFTAV RGLEGIKEAE VDIKGKKVRI AIANGIGNAK KLIEK IKSG ETKYDFVEVM ACPGGCMSGG GQPYTDDPEF RKKRMEGIYK NDRNLPKRKS HENEEVKKVY EEYYEKPCGP KAHEEL HTH YHSRKKEY

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Macromolecule #2: Electron bifurcating hydrogenase subunit HydB

MacromoleculeName: Electron bifurcating hydrogenase subunit HydB / type: protein_or_peptide / ID: 2 / Number of copies: 2 / Enantiomer: LEVO / EC number: hydrogen dehydrogenase (NADP+)
Source (natural)Organism: Thermoanaerobacter kivui (bacteria)
Molecular weightTheoretical: 68.279047 KDa
SequenceString: MVKLKSIQEL ENLREKIKEA KKKEKIVIRI CGGTGCRASG SLAVRDELVK VLKREGFANV DVNLSSDCLE NTSEVHVKMT GCQGFCAQG PLMTIEPLGV FYVGVKPEDV EEIVEKSIKK NEIIERLLYH DPATGKTYVK RDENPFYAKQ TRLVLKHCGT V DPASVYDY ...String:
MVKLKSIQEL ENLREKIKEA KKKEKIVIRI CGGTGCRASG SLAVRDELVK VLKREGFANV DVNLSSDCLE NTSEVHVKMT GCQGFCAQG PLMTIEPLGV FYVGVKPEDV EEIVEKSIKK NEIIERLLYH DPATGKTYVK RDENPFYAKQ TRLVLKHCGT V DPASVYDY IAEGGYSAIA KALTMDRKQI IDEVIKSGLR GRGGAGFPTG EKWLGAYKNQ SPKKYIICNG DEGDPGAFMD RS VMEGDPH KVIEGMMIGA YAIGSDEGYI YVRAEYPLAV QMLRKAIEEC EKLGLLGDNI LGTGFSFRLH VREGAGAFVC GES TALTYS IEGKRGMPRV RPPRTNECGL WEMPTVLNNV ETFACIPEII LNGGEWFASI GTPTSTGTKI FALSGKVNRT GLVE VPMGL KLRELIFDIG GGIANNKKFK AVQLGGPSGG CVPESQLDLP IDFDSLSKAG AIMGSGGVVV VDEDTCMVDF AKFFT NFIV EESCGKCIPC REGNKKMLEI LERITEGKGK EGDIELLEEL GDVIISASLC GLGKTAPNPV LSTIKHFRDE YEAHIR DKK CPAGACQALA AYKIDPGKCI GCGKCVKVCP VGAISGEKKK PHVIDQSKCI KCGACAENCP KGAIYKG

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Macromolecule #3: Electron bifurcating hydrogenase subunit HydC

MacromoleculeName: Electron bifurcating hydrogenase subunit HydC / type: protein_or_peptide / ID: 3 / Number of copies: 2 / Enantiomer: LEVO / EC number: hydrogen dehydrogenase (NADP+)
Source (natural)Organism: Thermoanaerobacter kivui (bacteria)
Molecular weightTheoretical: 18.624678 KDa
SequenceString:
MCNCCCKGSK DPRFEKVDEI LSKLANERGA LIAILQHVQH EFGYLPEDVI FYIASKTGIP ASKIYGVATF YAQFHLKPRG KYVIRVCLG TACHVKGANK ILAEFEKQLG IKAGETTSDL KFTLERVGCL GACGLAPTVM VNEKTYGKMT PEKVSEVLKE Y SDVEAAAS AQ

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Macromolecule #4: IRON/SULFUR CLUSTER

MacromoleculeName: IRON/SULFUR CLUSTER / type: ligand / ID: 4 / Number of copies: 14 / Formula: SF4
Molecular weightTheoretical: 351.64 Da
Chemical component information

ChemComp-FS1:
IRON/SULFUR CLUSTER / Iron–sulfur cluster

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Macromolecule #5: 2 IRON/2 SULFUR/5 CARBONYL/2 WATER INORGANIC CLUSTER

MacromoleculeName: 2 IRON/2 SULFUR/5 CARBONYL/2 WATER INORGANIC CLUSTER / type: ligand / ID: 5 / Number of copies: 2 / Formula: HC1
Molecular weightTheoretical: 355.933 Da
Chemical component information

ChemComp-HC1:
2 IRON/2 SULFUR/5 CARBONYL/2 WATER INORGANIC CLUSTER

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Macromolecule #6: FE2/S2 (INORGANIC) CLUSTER

MacromoleculeName: FE2/S2 (INORGANIC) CLUSTER / type: ligand / ID: 6 / Number of copies: 6 / Formula: FES
Molecular weightTheoretical: 175.82 Da
Chemical component information

ChemComp-FES:
FE2/S2 (INORGANIC) CLUSTER / Iron–sulfur cluster

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Macromolecule #7: FLAVIN MONONUCLEOTIDE

MacromoleculeName: FLAVIN MONONUCLEOTIDE / type: ligand / ID: 7 / Number of copies: 2 / Formula: FMN
Molecular weightTheoretical: 456.344 Da
Chemical component information

ChemComp-FMN:
FLAVIN MONONUCLEOTIDE / Flavin mononucleotide

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Macromolecule #8: ZINC ION

MacromoleculeName: ZINC ION / type: ligand / ID: 8 / Number of copies: 2 / Formula: ZN
Molecular weightTheoretical: 65.409 Da

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Macromolecule #9: NADP NICOTINAMIDE-ADENINE-DINUCLEOTIDE PHOSPHATE

MacromoleculeName: NADP NICOTINAMIDE-ADENINE-DINUCLEOTIDE PHOSPHATE / type: ligand / ID: 9 / Number of copies: 2 / Formula: NAP
Molecular weightTheoretical: 743.405 Da
Chemical component information

ChemComp-NAP:
NADP NICOTINAMIDE-ADENINE-DINUCLEOTIDE PHOSPHATE / Nicotinamide adenine dinucleotide phosphate

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Experimental details

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Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

BufferpH: 7.6
VitrificationCryogen name: ETHANE-PROPANE

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Electron microscopy

MicroscopeJEOL CRYO ARM 200
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Nominal defocus max: 2.0 µm / Nominal defocus min: 0.8 µm
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Average electron dose: 50.0 e/Å2

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Image processing

Initial angle assignmentType: PROJECTION MATCHING
Final angle assignmentType: PROJECTION MATCHING
Final reconstructionResolution.type: BY AUTHOR / Resolution: 3.1 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 193719

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