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- EMDB-13819: CryoEM structure of electron bifurcating Fe-Fe hydrogenase HydABC... -

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Basic information

Entry
Database: EMDB / ID: EMD-13819
TitleCryoEM structure of electron bifurcating Fe-Fe hydrogenase HydABC complex A. woodii in the oxidised state
Map dataSingle-Particle Cryo-EM Reconstructions of the Electron Bifurcating Hydrogenase HydABC symmetric(Processed on Cryosparc - non-sharpened map)
Sample
  • Complex: Homodimer complex of HydABC trimer
    • Protein or peptide: Iron hydrogenase HydA1
    • Protein or peptide: Iron hydrogenase HydB
    • Protein or peptide: Iron hydrogenase HydC
  • Ligand: IRON/SULFUR CLUSTER
  • Ligand: FE2/S2 (INORGANIC) CLUSTER
  • Ligand: FLAVIN MONONUCLEOTIDE
  • Ligand: ZINC ION
Keywordsflavin-based electron bifurcating hydrogenase / FeFe hydrogenase / nicotine amide adenine dinucleotide / ferredoxin / ELECTRON TRANSPORT
Function / homology
Function and homology information


ferredoxin hydrogenase / ferredoxin hydrogenase activity / NADH dehydrogenase (ubiquinone) activity / ATP synthesis coupled electron transport / FMN binding / 4 iron, 4 sulfur cluster binding / iron ion binding / membrane / metal ion binding
Similarity search - Function
: / 4Fe-4S dicluster 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 ...: / 4Fe-4S dicluster 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 / Soluble ligand binding domain / SLBB domain / : / NADH-quinone oxidoreductase subunit 3, ferredoxin-like 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 / 2Fe-2S iron-sulfur cluster binding domain / 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, iron-sulphur binding domain / NADH-ubiquinone oxidoreductase 51kDa subunit, iron-sulphur binding domain superfamily / NADH-ubiquinone oxidoreductase-F iron-sulfur binding region / NADH-ubiquinone oxidoreductase-F iron-sulfur binding region / NADH-ubiquinone oxidoreductase 51kDa subunit, FMN-binding domain / NADH-ubiquinone oxidoreductase 51kDa subunit, FMN-binding domain superfamily / Respiratory-chain NADH dehydrogenase 51 Kd subunit / Thioredoxin-like [2Fe-2S] ferredoxin / 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 / Prokaryotic membrane lipoprotein lipid attachment site profile. / Thioredoxin-like superfamily
Similarity search - Domain/homology
Iron hydrogenase HydA1 / Iron hydrogenase HydB
Similarity search - Component
Biological speciesAcetobacterium woodii DSM 1030 (bacteria)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.78 Å
AuthorsKumar A / Saura P / Poeverlein MC / Gamiz-Hernandez AP / Kaila VRI / Mueller V / Schuller JM
Funding support6 items
OrganizationGrant numberCountry
German Research Foundation (DFG)SCHU 3364/1-1
European Research Council (ERC)715311 (VRIK)
European Research Council (ERC)741791
Knut and Alice Wallenberg FoundationVRIK
German Research Foundation (DFG)SFB1078
Swedish Research Council016-07213
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
DepositionNov 2, 2021-
Header (metadata) releaseFeb 15, 2023-
Map releaseFeb 15, 2023-
UpdateJul 17, 2024-
Current statusJul 17, 2024Processing site: PDBe / Status: Released

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Structure visualization

Supplemental images

emd_13819.png

Downloads & links

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Map

FileDownload / File: emd_13819.map.gz / Format: CCP4 / Size: 30.5 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationSingle-Particle Cryo-EM Reconstructions of the Electron Bifurcating Hydrogenase HydABC symmetric(Processed on Cryosparc - non-sharpened map)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.09 Å/pix.
x 200 pix.
= 218. Å		1.09 Å/pix.
x 200 pix.
= 218. Å		1.09 Å/pix.
x 200 pix.
= 218. Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.09 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.0642
Minimum - Maximum-0.11923191 - 0.86848813
Average (Standard dev.)0.0023200526 (±0.020233475)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions200200200
Spacing200200200
CellA=B=C: 218.0 Å
α=β=γ: 90.0 °

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Supplemental data

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Additional map: Single-Particle Cryo-EM Reconstructions of the Electron Bifurcating Hydrogenase...

Fileemd_13819_additional_1.map
AnnotationSingle-Particle Cryo-EM Reconstructions of the Electron Bifurcating Hydrogenase HydABC symmetric(Processed on Cryosparc sharpened map)
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : Homodimer complex of HydABC trimer

EntireName: Homodimer complex of HydABC trimer
Components
  • Complex: Homodimer complex of HydABC trimer
    • Protein or peptide: Iron hydrogenase HydA1
    • Protein or peptide: Iron hydrogenase HydB
    • Protein or peptide: Iron hydrogenase HydC
  • Ligand: IRON/SULFUR CLUSTER
  • Ligand: FE2/S2 (INORGANIC) CLUSTER
  • Ligand: FLAVIN MONONUCLEOTIDE
  • Ligand: ZINC ION

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Supramolecule #1: Homodimer complex of HydABC trimer

SupramoleculeName: Homodimer complex of HydABC trimer / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#3
Source (natural)Organism: Acetobacterium woodii DSM 1030 (bacteria)
Molecular weightTheoretical: 350 kDa/nm

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Macromolecule #1: Iron hydrogenase HydA1

MacromoleculeName: Iron hydrogenase HydA1 / type: protein_or_peptide / ID: 1 / Number of copies: 2 / Enantiomer: LEVO / EC number: ferredoxin hydrogenase
Source (natural)Organism: Acetobacterium woodii DSM 1030 (bacteria)
Molecular weightTheoretical: 63.692887 KDa
SequenceString: MKEITFKING QEMIVPEGTT ILEAARMNNI DIPTLCYLKD INEIGACRMC LVEIAGARAL QAACVYPVAN GIEVLTNSPK VREARRVNL ELILSNHNRE CTTCIRSENC ELQTLATDLG VSDIPFEGEK SGKLIDDLST SVVRDESKCI LCKRCVSVCR D VQSVAVLG ...String:
MKEITFKING QEMIVPEGTT ILEAARMNNI DIPTLCYLKD INEIGACRMC LVEIAGARAL QAACVYPVAN GIEVLTNSPK VREARRVNL ELILSNHNRE CTTCIRSENC ELQTLATDLG VSDIPFEGEK SGKLIDDLST SVVRDESKCI LCKRCVSVCR D VQSVAVLG TVGRGFTSQV QPVFNKSLAD VGCINCGQCI INCPVGALKE KSDIQRVWDA IADPSKTVIV QTAPAVRAAL GE EFGYPMG TSVTGKMAAA LRRLGFDKVF DTDFGADVCI MEEGTELIGR VTNGGVLPMI TSCSPGWIKF IETYYPEAIP HLS SCKSPQ NITGALLKNH YAQTNNIDPK DMVVVSIMPC TAKKYEVQRE ELCTDGNADV DISITTRELA RMIKEARILF NKLP DEDFD DYYGESTGAA VIFGATGGVM EAAVRTVADV LNKKDIQEID YQIVRGVDGI KKASVEVTPD LTVNLVVAHG GANIR EVME QLKAGELADT HFIELMACPG GCVNGGGQPI VSAKDKMDID IRTERAKALY DEDANVLTYR KSHQNPSVIR LYEEYL EEP NSPKAHHILH TKYSAKPKLV

UniProtKB: Iron hydrogenase HydA1

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Macromolecule #2: Iron hydrogenase HydB

MacromoleculeName: Iron hydrogenase HydB / type: protein_or_peptide / ID: 2 / Number of copies: 2 / Enantiomer: LEVO / EC number: ferredoxin hydrogenase
Source (natural)Organism: Acetobacterium woodii DSM 1030 (bacteria) / Strain: ATCC 29683 / DSM 1030 / JCM 2381 / KCTC 1655 / WB1
Molecular weightTheoretical: 50.264406 KDa
SequenceString: NIDEYIGFDG YLALEKVLLT MSPVDVINEV KASGLRGRGG GGFPTGLKWQ FAHDAVSEDG IKYVACNADE GDPGAFMDRS VLEGDPHAV IEAMAIAGYA VGASKGYVYV RAEYPIAVNR LQIAIDQAKE YGILGENIFE TDFSFDLEIR LGAGAFVCGE E TALMNSIE ...String:
NIDEYIGFDG YLALEKVLLT MSPVDVINEV KASGLRGRGG GGFPTGLKWQ FAHDAVSEDG IKYVACNADE GDPGAFMDRS VLEGDPHAV IEAMAIAGYA VGASKGYVYV RAEYPIAVNR LQIAIDQAKE YGILGENIFE TDFSFDLEIR LGAGAFVCGE E TALMNSIE GKRGEPRPRP PFPANKGLFG KPTVLNNVET YANIPKIILN GAEWFASVGT EKSKGTKVFA LGGKINNTGL LE IPMGTTL REIIYEIGGG IPNGKAFKAA QTGGPSGGCL PESLLDTEID YDNLIAAGSM MGSGGLIVMD EDNCMVDVAR FFL DFTQDE SCGKCPPCRI GTKRMLEILE RICDGKGVEG DIERLEELAV GIKSSALCGL GQTAPNPVLS TIRFFRDEYE AHIR DKKCP AGVCKHLLDF KINADTCKGC GICAKKCPAD AISGEKKKPY NIDTSKCIKC GACIEACPFG SISKA

UniProtKB: Iron hydrogenase HydB

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Macromolecule #3: Iron hydrogenase HydC

MacromoleculeName: Iron hydrogenase HydC / type: protein_or_peptide / ID: 3 / Number of copies: 2 / Enantiomer: LEVO / EC number: ferredoxin hydrogenase
Source (natural)Organism: Acetobacterium woodii DSM 1030 (bacteria)
Molecular weightTheoretical: 16.999689 KDa
SequenceString:
MAELIPVENL DVVKAIVAEH REVPGCLMQI LQETQLKYGY LPLELQGTIA DELGIPLTEV YGVATFYSQF TLKPKGKYKI GICLGTACY VRGSQAIIDK VNSVLGTQVG DTTEDGKWSV DATRCVGACG LAPVMMINEE VFGRLTVDEI PGILEKY

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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

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Macromolecule #5: FE2/S2 (INORGANIC) CLUSTER

MacromoleculeName: FE2/S2 (INORGANIC) CLUSTER / type: ligand / ID: 5 / Number of copies: 4 / Formula: FES
Molecular weightTheoretical: 175.82 Da
Chemical component information

ChemComp-FES:
FE2/S2 (INORGANIC) CLUSTER

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Macromolecule #6: FLAVIN MONONUCLEOTIDE

MacromoleculeName: FLAVIN MONONUCLEOTIDE / type: ligand / ID: 6 / Number of copies: 2 / Formula: FMN
Molecular weightTheoretical: 456.344 Da
Chemical component information

ChemComp-FMN:
FLAVIN MONONUCLEOTIDE

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Macromolecule #7: ZINC ION

MacromoleculeName: ZINC ION / type: ligand / ID: 7 / Number of copies: 2 / Formula: ZN
Molecular weightTheoretical: 65.409 Da

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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

MicroscopeFEI TITAN KRIOS
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Average electron dose: 55.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Nominal defocus max: 2.0 µm / Nominal defocus min: 0.8 µm
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Startup modelType of model: INSILICO MODEL
Final reconstructionResolution.type: BY AUTHOR / Resolution: 3.78 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 250709
Initial angle assignmentType: PROJECTION MATCHING
Final angle assignmentType: PROJECTION MATCHING

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