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- EMDB-60383: Anaerobically isolated active [FeFe]-hydrogenase CbA5H -

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Basic information

Entry
Database: EMDB / ID: EMD-60383
TitleAnaerobically isolated active [FeFe]-hydrogenase CbA5H
Map data
Sample
  • Complex: homodimeric structure of the active [FeFe]-hydrogenase CbA5H
    • Protein or peptide: [FeFe]-hydrogenase
  • Ligand: ZINC ION
  • Ligand: IRON/SULFUR CLUSTER
  • Ligand: dicarbonyl[bis(cyanide-kappaC)]-mu-(iminodimethanethiolatato-1kappaS:2kappaS)-mu-(oxomethylidene)diiron(2+)
Keywords[FeFe]-hydrogenases / homodimer / Zn-binding / OXIDOREDUCTASE
Function / homology
Function and homology information


ferredoxin hydrogenase activity / 4 iron, 4 sulfur cluster binding / iron 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 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 / 4Fe-4S dicluster domain / Soluble ligand binding domain / SLBB domain / 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
Similarity search - Domain/homology
Biological speciesClostridium beijerinckii (bacteria)
Methodsingle particle reconstruction / cryo EM / Resolution: 2.21 Å
AuthorsKawamoto A / Kurisu G
Funding support Germany, Japan, 6 items
OrganizationGrant numberCountry
German Research Foundation (DFG)EXC2033-390677874-RESOLV Germany
Japan Science and TechnologyJPMJSC21C2 Japan
German Research Foundation (DFG)HA2555/10-1 Germany
German Research Foundation (DFG)AP242/12-1 Germany
Japan Society for the Promotion of Science (JSPS)21H02417 Japan
German Research Foundation (DFG)461338801 Germany
CitationJournal: Proc Natl Acad Sci U S A / Year: 2025
Title: Structural determinants of oxygen resistance and Zn-mediated stability of the [FeFe]-hydrogenase from .
Authors: Jifu Duan / Andreas Rutz / Akihiro Kawamoto / Shuvankar Naskar / Kristina Edenharter / Silke Leimkühler / Eckhard Hofmann / Thomas Happe / Genji Kurisu /
Abstract: [FeFe]-hydrogenases catalyze the reversible two-electron reduction of two protons to molecular hydrogen. Although these enzymes are among the most efficient H-converting biocatalysts in nature, their ...[FeFe]-hydrogenases catalyze the reversible two-electron reduction of two protons to molecular hydrogen. Although these enzymes are among the most efficient H-converting biocatalysts in nature, their catalytic cofactor (termed H-cluster) is irreversibly destroyed upon contact with dioxygen. The [FeFe]-hydrogenase CbA5H from has a unique mechanism to protect the H-cluster from oxygen-induced degradation. The protective strategy of CbA5H was proposed based on a partial protein structure of CbA5H's oxygen-shielded form. Here, we present a cryo-EM structure of 2.2 Å resolution from the entire enzyme in its dimeric and active state and elucidate the structural parameters of the reversible cofactor protection mechanism. We found that both subunits of the homodimeric structure of CbA5H have a Zn-binding four-helix domain, which does not play a role in electron transport as described for other complex protein structures. Biochemical data instead confirm that two [4Fe-4S] clusters are responsible for electron transfer in CbA5H, while the identified zinc atom is critical for oligomerization and protein stability.
History
DepositionJun 1, 2024-
Header (metadata) releaseJan 1, 2025-
Map releaseJan 1, 2025-
UpdateJul 16, 2025-
Current statusJul 16, 2025Processing site: PDBj / Status: Released

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Structure visualization

Supplemental images

emd_60383.png

Downloads & links

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Map

FileDownload / File: emd_60383.map.gz / Format: CCP4 / Size: 107.2 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
0.68 Å/pix.
x 304 pix.
= 205.2 Å		0.68 Å/pix.
x 304 pix.
= 205.2 Å		0.68 Å/pix.
x 304 pix.
= 205.2 Å

Surface

Projections

Slices (1/3)

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Images are generated by Spider.

Voxel sizeX=Y=Z: 0.675 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.0176
Minimum - Maximum-0.080237314 - 0.17372502
Average (Standard dev.)0.00012878688 (±0.0036475668)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions304304304
Spacing304304304
CellA=B=C: 205.2 Å
α=β=γ: 90.0 °

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Supplemental data

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Mask #1

Fileemd_60383_msk_1.map
Projections & Slices
AxesZYX

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Additional map: #1

Fileemd_60383_additional_1.map
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Half map: #2

Fileemd_60383_half_map_1.map
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Half map: #1

Fileemd_60383_half_map_2.map
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Sample components

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Entire : homodimeric structure of the active [FeFe]-hydrogenase CbA5H

EntireName: homodimeric structure of the active [FeFe]-hydrogenase CbA5H
Components
  • Complex: homodimeric structure of the active [FeFe]-hydrogenase CbA5H
    • Protein or peptide: [FeFe]-hydrogenase
  • Ligand: ZINC ION
  • Ligand: IRON/SULFUR CLUSTER
  • Ligand: dicarbonyl[bis(cyanide-kappaC)]-mu-(iminodimethanethiolatato-1kappaS:2kappaS)-mu-(oxomethylidene)diiron(2+)

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Supramolecule #1: homodimeric structure of the active [FeFe]-hydrogenase CbA5H

SupramoleculeName: homodimeric structure of the active [FeFe]-hydrogenase CbA5H
type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1
Source (natural)Organism: Clostridium beijerinckii (bacteria)

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Macromolecule #1: [FeFe]-hydrogenase

MacromoleculeName: [FeFe]-hydrogenase / type: protein_or_peptide / ID: 1 / Number of copies: 2 / Enantiomer: LEVO
Source (natural)Organism: Clostridium beijerinckii (bacteria)
Molecular weightTheoretical: 75.001938 KDa
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString: MGDNKKSFIQ SALGSVFSVF SEEELKELSN GRKIAICGKV NNPGIIEVPE GATLNEIIQL CGGLINKSNF KAAQIGLPFG GFLTEDSLD KEFDFGIFYE NIARTIIVLS QEDCIIQFEK FYIEYLLAKI KDGSYKNYEV VKEDITEMFN ILNRISKGVS N MREIYLLR ...String:
MGDNKKSFIQ SALGSVFSVF SEEELKELSN GRKIAICGKV NNPGIIEVPE GATLNEIIQL CGGLINKSNF KAAQIGLPFG GFLTEDSLD KEFDFGIFYE NIARTIIVLS QEDCIIQFEK FYIEYLLAKI KDGSYKNYEV VKEDITEMFN ILNRISKGVS N MREIYLLR NLAVTVKSKM NQKHNIMEEI IDKFYEEIEE HIEEKKCYTS QCNHLVKLTI TKKCIGCGAC KRACPVDCIN GE LKKKHEI DYNRCTHCGA CVSACPVDAI SAGDNTMLFL RDLATPNKVV ITQMAPAVRV AIGEAFGFEP GENVEKKIAA GLR KLGVDY VFDTSWGADL TIMEEAAELQ ERLERHLAGD ESVKLPILTS CCPSWIKFIE QNYGDMLDVP SSAKSPMEMF AIVA KEIWA KEKGLSRDEV TSVAIMPCIA KKYEASRAEF SVDMNYDVDY VITTRELIKI FENSGINLKE IEDEEIDTVM GEYTG AGII FGRTGGVIEA ATRTALEKMT GERFDNIEFE GLRGWDGFRV CELEAGDIKL RIGVAHGLRE AAKMLDKIRS GEEFFH AIE IMACVGGCIG GGGQPKTKGN KQAALQKRAE GLNNIDRSKT LRRSNENPEV LAIYEKYLDH PLSNKAHELL HTVYFPR VK KDDIWSVGVK LFGGGSGGGS GGGSWSHPQF EK

UniProtKB: [FeFe]-hydrogenase

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Macromolecule #2: ZINC ION

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

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Macromolecule #3: IRON/SULFUR CLUSTER

MacromoleculeName: IRON/SULFUR CLUSTER / type: ligand / ID: 3 / Number of copies: 6 / Formula: SF4
Molecular weightTheoretical: 351.64 Da
Chemical component information

ChemComp-FS1:
IRON/SULFUR CLUSTER

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Macromolecule #4: dicarbonyl[bis(cyanide-kappaC)]-mu-(iminodimethanethiolatato-1kap...

MacromoleculeName: dicarbonyl[bis(cyanide-kappaC)]-mu-(iminodimethanethiolatato-1kappaS:2kappaS)-mu-(oxomethylidene)diiron(2+)
type: ligand / ID: 4 / Number of copies: 2 / Formula: 402
Molecular weightTheoretical: 354.953 Da
Chemical component information

ChemComp-402:
dicarbonyl[bis(cyanide-kappaC)]-mu-(iminodimethanethiolatato-1kappaS:2kappaS)-mu-(oxomethylidene)diiron(2+)

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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: 8
Component:
ConcentrationFormulaName
100.0 mMTris-HClTris Hydrochloride
2.0 mMNaDTsodium dithionite

Details: 100 mM Tris-HCl, 2mM NaDT
GridModel: Quantifoil R1.2/1.3 / Material: COPPER / Mesh: 200 / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 30 sec.
VitrificationCryogen name: ETHANE / Chamber humidity: 50 % / Chamber temperature: 278 K / Instrument: FEI VITROBOT MARK IV / Details: Vitrification carried out in anaerobic condition.

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Specialist opticsSpherical aberration corrector: Microscope was modified with a Cs corrector
Energy filter - Name: GIF Bioquantum / Energy filter - Slit width: 20 eV
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Number grids imaged: 1 / Number real images: 9994 / Average electron dose: 60.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 50.0 µm / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 0.01 mm / Nominal defocus max: 2.0 µm / Nominal defocus min: 0.6 µm / Nominal magnification: 105000
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 7759751
CTF correctionSoftware - Name: Gctf / Type: PHASE FLIPPING ONLY
Startup modelType of model: INSILICO MODEL
Final reconstructionApplied symmetry - Point group: C2 (2 fold cyclic) / Resolution.type: BY AUTHOR / Resolution: 2.21 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 3.1) / Number images used: 175586
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 3.1)
Final angle assignmentType: ANGULAR RECONSTITUTION / Software - Name: RELION (ver. 3.1)
Final 3D classificationNumber classes: 10 / Software - Name: RELION (ver. 3.1)
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial modelPDB ID:

6ttl


Chain - Source name: PDB / Chain - Initial model type: experimental model
RefinementSpace: REAL / Protocol: FLEXIBLE FIT
Output model

PDB-8zqd:
Anaerobically isolated active [FeFe]-hydrogenase CbA5H

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