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- PDB-28fu: Cryo-EM structure of hydrogenase-2 in complex with L1 (right) -

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Basic information

Entry
Database: PDB / ID: 28fu
TitleCryo-EM structure of hydrogenase-2 in complex with L1 (right)
Components
  • (Hydrogenase-2 ...) x 2
  • binder L1
KeywordsOXIDOREDUCTASE / Complex / Binder
Function / homology
Function and homology information


hydrogenase (acceptor) / ferredoxin hydrogenase complex / [Ni-Fe] hydrogenase complex / hydrogenase (acceptor) activity / ferredoxin hydrogenase activity / anaerobic respiration / nickel cation binding / iron-sulfur cluster binding / electron transfer activity / periplasmic space ...hydrogenase (acceptor) / ferredoxin hydrogenase complex / [Ni-Fe] hydrogenase complex / hydrogenase (acceptor) activity / ferredoxin hydrogenase activity / anaerobic respiration / nickel cation binding / iron-sulfur cluster binding / electron transfer activity / periplasmic space / membrane / plasma membrane
Similarity search - Function
: / [NiFe]-hydrogenase, small subunit / Cytochrome-c3 hydrogenase, C-terminal / [NiFe]-hydrogenase, small subunit, C-terminal domain superfamily / NiFe/NiFeSe hydrogenase small subunit C-terminal / HoxL-like / Nickel-dependent hydrogenases large subunit signature 2. / Nickel-dependent hydrogenases large subunit signature 1. / [NiFe]-hydrogenase, small subunit, N-terminal domain superfamily / Nickel-dependent hydrogenase, large subunit, nickel binding site ...: / [NiFe]-hydrogenase, small subunit / Cytochrome-c3 hydrogenase, C-terminal / [NiFe]-hydrogenase, small subunit, C-terminal domain superfamily / NiFe/NiFeSe hydrogenase small subunit C-terminal / HoxL-like / Nickel-dependent hydrogenases large subunit signature 2. / Nickel-dependent hydrogenases large subunit signature 1. / [NiFe]-hydrogenase, small subunit, N-terminal domain superfamily / Nickel-dependent hydrogenase, large subunit, nickel binding site / Nickel-dependent hydrogenase, large subunit / Nickel-dependent hydrogenase / Twin-arginine translocation pathway, signal sequence, bacterial/archaeal / NADH:ubiquinone oxidoreductase-like, 20kDa subunit / NADH ubiquinone oxidoreductase, 20 Kd subunit / [NiFe]-hydrogenase, large subunit / Twin arginine translocation (Tat) signal profile. / Twin-arginine translocation pathway, signal sequence
Similarity search - Domain/homology
FE3-S4 CLUSTER / CARBONMONOXIDE-(DICYANO) IRON / NICKEL (II) ION / IRON/SULFUR CLUSTER / Hydrogenase-2 large chain / Hydrogenase-2 small chain
Similarity search - Component
Biological speciessynthetic construct (others)
Escherichia coli (E. coli)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.48 Å
AuthorsZhang, N. / Huang, Y. / Zhang, L.
Funding support China, 1items
OrganizationGrant numberCountry
Ministry of Science and Technology (MoST, China)2020YFA0907300 China
CitationJournal: Angew Chem Int Ed Engl / Year: 2026
Title: De Novo Design of a Protein Binder to Probe Gas Channel and Enhance the Oxygen Tolerance of [NiFe]-Hydrogenase.
Authors: Xuan Sun / Qi Xiao / Nan Zhang / Wenjin Li / Hang Luo / Yanxin Gao / Leyan Zhang / Yilin Fan / Peiyu Jiang / Yucen Huang / Geng Wu / Liyun Zhang /
Abstract: [NiFe]-Hydrogenases display remarkable catalytic efficiency for H production and oxidation but are sensitive to O, which severely restricts their biotechnological applications. The identities and ...[NiFe]-Hydrogenases display remarkable catalytic efficiency for H production and oxidation but are sensitive to O, which severely restricts their biotechnological applications. The identities and functional relevance of gas channels leading to the buried [NiFe] active site remain elusive, hindering rational enzyme engineering. Here, we introduce an artificial intelligence-guided de novo protein binder design strategy to map hydrophobic O diffusion pathways in Escherichia coli hydrogenase-2 (Hyd-2). By integrating RFdiffusion, ProteinMPNN, and AlphaFold, ∼100,000 candidate binders were computationally screened, yielding two high-affinity binders, L1 and L2. Biophysical and electrochemical analyses show that L1 selectively occludes the primary O ingress channel, enhancing the enzyme's oxygen tolerance by more than threefold, whereas L2, which targets another putative channel, has a negligible effect, indicating that this pathway contributes minimally to O ingress under the tested conditions. Furthermore, we determined the L1-Hyd-2 complex structure by cryo-electron microscopy and revealed the key interaction residues and interface conformation. Integrated structural and computational analyses provide mechanistic insights into the system's oxygen tolerance and high-affinity L1-Hyd-2 interaction. All findings provide the first direct experimental evidence for hierarchical O diffusion channels in [NiFe]-hydrogenases and establish binder-mediated channel occlusion as a generalizable, mutation-free strategy for elucidating and modulating gas transport in metalloenzymes.
History
DepositionJun 24, 2026Deposition site: PDBJ / Processing site: PDBC
Revision 1.0Oct 14, 2026Provider: repository / Type: Initial release
Revision 1.0Oct 14, 2026Data content type: EM metadata / Data content type: EM metadata / Provider: repository / Type: Initial release

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
E: binder L1
A: Hydrogenase-2 small chain
B: Hydrogenase-2 large chain
C: Hydrogenase-2 small chain
D: Hydrogenase-2 large chain
hetero molecules


Theoretical massNumber of molelcules
Total (without water)200,60215
Polymers198,2155
Non-polymers2,38710
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: electron microscopy, not applicable
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

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Protein , 1 types, 1 molecules E

#1: Protein binder L1


Mass: 8356.343 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) synthetic construct (others) / Production host: Escherichia coli (E. coli)

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Hydrogenase-2 ... , 2 types, 4 molecules ACBD

#2: Protein Hydrogenase-2 small chain / HYD2 / Membrane-bound hydrogenase 2 small subunit / NiFe hydrogenase


Mass: 32371.498 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Escherichia coli (strain K12) (bacteria)
Strain: K12 / Gene: hybO, yghV, b2997, JW2965 / Production host: Escherichia coli (E. coli) / References: UniProt: P69741, hydrogenase (acceptor)
#3: Protein Hydrogenase-2 large chain / HYD2 / Membrane-bound hydrogenase 2 large subunit / NiFe hydrogenase


Mass: 62557.906 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Escherichia coli (strain K12) (bacteria)
Strain: K12 / Gene: hybC, b2994, JW2962 / Production host: Escherichia coli (E. coli) / References: UniProt: P0ACE0, hydrogenase (acceptor)

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Non-polymers , 4 types, 10 molecules

#4: Chemical
ChemComp-SF4 / IRON/SULFUR CLUSTER


Mass: 351.640 Da / Num. of mol.: 4 / Source method: obtained synthetically / Formula: Fe4S4
#5: Chemical ChemComp-F3S / FE3-S4 CLUSTER


Mass: 295.795 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: Fe3S4
#6: Chemical ChemComp-FCO / CARBONMONOXIDE-(DICYANO) IRON


Mass: 135.890 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: C3FeN2O
#7: Chemical ChemComp-NI / NICKEL (II) ION


Mass: 58.693 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: Ni

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Details

Has ligand of interestN
Has protein modificationY

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction

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Sample preparation

ComponentName: The complex of hydrogenase-2 with binder L1 (right) / Type: COMPLEX / Entity ID: #1-#3 / Source: RECOMBINANT
Source (natural)Organism: Escherichia coli (strain K12) (bacteria)
Source (recombinant)Organism: Escherichia coli (E. coli)
Buffer solutionpH: 7.2
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
VitrificationCryogen name: ETHANE

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Electron microscopy imaging

MicroscopyModel: TFS TITAN THEMIS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal defocus max: 2000 nm / Nominal defocus min: 800 nm
Image recordingElectron dose: 50 e/Å2 / Film or detector model: FEI FALCON IV (4k x 4k)

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Processing

EM software
IDNameVersionCategory
1cryoSPARCparticle selection
2PHENIX1.20.1_4487model refinement
13cryoSPARC3D reconstruction
CTF correctionType: NONE
3D reconstructionResolution: 2.48 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 241696 / Symmetry type: POINT
RefinementHighest resolution: 2.48 Å
Stereochemistry target values: REAL-SPACE (WEIGHTED MAP SUM AT ATOM CENTERS)
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00913688
ELECTRON MICROSCOPYf_angle_d1.00318714
ELECTRON MICROSCOPYf_dihedral_angle_d3.5851867
ELECTRON MICROSCOPYf_chiral_restr0.0462062
ELECTRON MICROSCOPYf_plane_restr0.0052428

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