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- PDB-9nf0: Structure of the NADPH-bound Pyrococcus furiosus SHI complex -

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Basic information

Entry
Database: PDB / ID: 9nf0
TitleStructure of the NADPH-bound Pyrococcus furiosus SHI complex
Components(Sulfhydrogenase 1 subunit ...) x 4
KeywordsELECTRON TRANSPORT / hydrogen production / [NiFe] hydrogenase / cryo-EM structure
Function / homology
Function and homology information


sulfhydrogenase / sulfur reductase activity / hydrogen dehydrogenase (NADP+) / hydrogen dehydrogenase (NADP+) activity / [Ni-Fe] hydrogenase complex / pyrimidine nucleotide biosynthetic process / ferredoxin hydrogenase activity / 3 iron, 4 sulfur cluster binding / nickel cation binding / 2 iron, 2 sulfur cluster binding ...sulfhydrogenase / sulfur reductase activity / hydrogen dehydrogenase (NADP+) / hydrogen dehydrogenase (NADP+) activity / [Ni-Fe] hydrogenase complex / pyrimidine nucleotide biosynthetic process / ferredoxin hydrogenase activity / 3 iron, 4 sulfur cluster binding / nickel cation binding / 2 iron, 2 sulfur cluster binding / flavin adenine dinucleotide binding / 4 iron, 4 sulfur cluster binding / metal ion binding / cytoplasm
Similarity search - Function
: / : / 4Fe-4S dicluster domain / : / : / Dihydroorotate dehydrogenase, electron transfer subunit, Fe-S binding domain superfamily / Cytochrome-c3 hydrogenase, gamma subunit / Dihydroorotate dehydrogenase, electron transfer subunit, iron-sulphur cluster binding domain / : / Iron-sulfur cluster binding domain of dihydroorotate dehydrogenase B ...: / : / 4Fe-4S dicluster domain / : / : / Dihydroorotate dehydrogenase, electron transfer subunit, Fe-S binding domain superfamily / Cytochrome-c3 hydrogenase, gamma subunit / Dihydroorotate dehydrogenase, electron transfer subunit, iron-sulphur cluster binding domain / : / Iron-sulfur cluster binding domain of dihydroorotate dehydrogenase B / : / Flavoprotein pyridine nucleotide cytochrome reductase-like, FAD-binding domain / Oxidoreductase FAD-binding domain / Nickel-dependent hydrogenases large subunit signature 2. / [NiFe]-hydrogenase, small subunit, N-terminal domain superfamily / Nickel-dependent hydrogenase, large subunit, nickel binding site / Nickel-dependent hydrogenase, large subunit / Nickel-dependent hydrogenase / Alpha-helical ferredoxin / NADH:ubiquinone oxidoreductase-like, 20kDa subunit / NADH ubiquinone oxidoreductase, 20 Kd subunit / [NiFe]-hydrogenase, large subunit / Oxidoreductase FAD/NAD(P)-binding / Oxidoreductase NAD-binding domain / 4Fe-4S ferredoxin, iron-sulphur binding, conserved site / 4Fe-4S ferredoxin-type iron-sulfur binding region signature. / FAD-binding domain, ferredoxin reductase-type / Ferredoxin-NADP reductase (FNR), nucleotide-binding domain / Ferredoxin reductase-type FAD binding domain profile. / Riboflavin synthase-like beta-barrel / 4Fe-4S ferredoxin-type iron-sulfur binding domain profile. / 4Fe-4S ferredoxin-type, iron-sulphur binding domain
Similarity search - Domain/homology
FLAVIN-ADENINE DINUCLEOTIDE / FE2/S2 (INORGANIC) CLUSTER / NADP NICOTINAMIDE-ADENINE-DINUCLEOTIDE PHOSPHATE / Chem-NFU / IRON/SULFUR CLUSTER / Sulfhydrogenase 1 subunit delta / Sulfhydrogenase 1 subunit alpha / Sulfhydrogenase 1 subunit gamma / Sulfhydrogenase 1 subunit beta
Similarity search - Component
Biological speciesPyrococcus furiosus (archaea)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.06 Å
AuthorsXiao, X. / Li, H.
Funding support United States, 1items
OrganizationGrant numberCountry
Department of Energy (DOE, United States) United States
CitationJournal: Structure / Year: 2025
Title: Structural insights into the biotechnologically relevant reversible NADPH-oxidizing NiFe-hydrogenase from P.furiosus.
Authors: Xiansha Xiao / Gerrit J Schut / Xiang Feng / Patrick M McTernan / Dominik K Haja / William N Lanzilotta / Michael W W Adams / Huilin Li /
Abstract: The cytoplasmic hydrogenase I (SHI) from the hyperthermophilic archaeon Pyrococcus furiosus belongs to the group III hydrogenase family. SHI oxidizes NADPH rather than NADH to reduce protons and ...The cytoplasmic hydrogenase I (SHI) from the hyperthermophilic archaeon Pyrococcus furiosus belongs to the group III hydrogenase family. SHI oxidizes NADPH rather than NADH to reduce protons and evolve hydrogen gas, and because of this property, coupled with its high thermal stability, the enzyme holds great potential for economical hydrogen production. Despite decades of efforts, the SHI structure has remained unknown. Here, we report the cryoelectron microscopic (cryo-EM) structures of the heterotetrameric SHI holoenzyme (αδβγ). SHI is a symmetric dimer of two individually functional heterotetramers. SHI-αδ resembles the standard [NiFe] hydrogenase, and SHI-βγ function as the NADPH oxidoreductase. SHI-β contains three [4Fe-4S] clusters that relay electrons from NADPH in SHI-γ to the catalytic [NiFe] cluster in SHI-αδ for H production. These structures will guide the adaptation of this unique enzyme for biotechnological applications.
History
DepositionFeb 20, 2025Deposition site: RCSB / Processing site: RCSB
Revision 1.0Jul 9, 2025Provider: repository / Type: Initial release
Revision 1.0Jul 9, 2025Data content type: EM metadata / Data content type: EM metadata / Provider: repository / Type: Initial release
Revision 1.0Jul 9, 2025Data content type: FSC / Data content type: FSC / Provider: repository / Type: Initial release
Revision 1.0Jul 9, 2025Data content type: Image / Data content type: Image / Provider: repository / Type: Initial release
Revision 1.0Jul 9, 2025Data content type: Primary map / Data content type: Primary map / Provider: repository / Type: Initial release
Revision 1.1Jul 16, 2025Group: Data collection / Source and taxonomy / Structure summary
Category: em_admin / em_entity_assembly ...em_admin / em_entity_assembly / em_entity_assembly_recombinant / entity / entity_src_gen / entity_src_nat
Item: _em_admin.last_update / _em_entity_assembly.source / _entity.src_method
Revision 1.1Jul 16, 2025Data content type: EM metadata / Data content type: EM metadata / EM metadata / EM metadata
Group: Experimental summary / Source and taxonomy / Structure summary
Data content type: EM metadata / EM metadata ...EM metadata / EM metadata / EM metadata / EM metadata / EM metadata / EM metadata
Category: em_admin / em_entity_assembly ...em_admin / em_entity_assembly / em_entity_assembly_recombinant / entity / entity_src_gen / entity_src_nat
Data content type: EM metadata / EM metadata / EM metadata
Item: _em_admin.last_update / _em_entity_assembly.source / _entity.src_method
Revision 1.2Sep 17, 2025Group: Data collection / Database references / Category: citation / em_admin
Item: _citation.journal_volume / _citation.page_first / _em_admin.last_update

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Sulfhydrogenase 1 subunit delta
B: Sulfhydrogenase 1 subunit beta
C: Sulfhydrogenase 1 subunit gamma
D: Sulfhydrogenase 1 subunit alpha
E: Sulfhydrogenase 1 subunit delta
F: Sulfhydrogenase 1 subunit beta
G: Sulfhydrogenase 1 subunit gamma
H: Sulfhydrogenase 1 subunit alpha
hetero molecules


Theoretical massNumber of molelcules
Total (without water)317,19132
Polymers308,4188
Non-polymers8,77224
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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Sulfhydrogenase 1 subunit ... , 4 types, 8 molecules AEBFCGDH

#1: Protein Sulfhydrogenase 1 subunit delta / Hydrogenase I small subunit / NADP-reducing hydrogenase subunit HydD / Sulfhydrogenase I subunit delta


Mass: 29269.037 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Pyrococcus furiosus (archaea) / Gene: hydD, PF0893 / Production host: Pyrococcus furiosus (archaea) / References: UniProt: E7FHU4, hydrogen dehydrogenase (NADP+)
#2: Protein Sulfhydrogenase 1 subunit beta / Sulfhydrogenase I subunit beta / Sulfur reductase subunit HydB


Mass: 43464.688 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Pyrococcus furiosus (archaea) / Gene: hydB, PF0891 / Production host: Pyrococcus furiosus (archaea) / References: UniProt: Q8U2E5, sulfhydrogenase
#3: Protein Sulfhydrogenase 1 subunit gamma / Sulfhydrogenase I subunit gamma / Sulfur reductase subunit HydG


Mass: 33093.527 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Pyrococcus furiosus (archaea) / Gene: hydG, PF0892 / Production host: Pyrococcus furiosus (archaea) / References: UniProt: Q8U2E4, sulfhydrogenase
#4: Protein Sulfhydrogenase 1 subunit alpha / Hydrogenase I large subunit / NADP-reducing hydrogenase subunit HydA / Sulfhydrogenase I subunit alpha


Mass: 48381.871 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Pyrococcus furiosus (archaea) / Gene: hydA, PF0894 / Production host: Pyrococcus furiosus (archaea) / References: UniProt: E7FI44, hydrogen dehydrogenase (NADP+)

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Non-polymers , 6 types, 24 molecules

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


Mass: 351.640 Da / Num. of mol.: 14 / Source method: obtained synthetically / Formula: Fe4S4 / Feature type: SUBJECT OF INVESTIGATION
#6: Chemical ChemComp-FES / FE2/S2 (INORGANIC) CLUSTER


Mass: 175.820 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: Fe2S2 / Feature type: SUBJECT OF INVESTIGATION
#7: Chemical ChemComp-FAD / FLAVIN-ADENINE DINUCLEOTIDE


Mass: 785.550 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: C27H33N9O15P2 / Feature type: SUBJECT OF INVESTIGATION / Comment: FAD*YM
#8: Chemical ChemComp-NAP / NADP NICOTINAMIDE-ADENINE-DINUCLEOTIDE PHOSPHATE / 2'-MONOPHOSPHOADENOSINE 5'-DIPHOSPHORIBOSE


Mass: 743.405 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: C21H28N7O17P3 / Feature type: SUBJECT OF INVESTIGATION
#9: Chemical ChemComp-NFU / formyl[bis(hydrocyanato-1kappaC)]ironnickel(Fe-Ni) / NI-FE REDUCED ACTIVE CENTER


Mass: 195.591 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: C3HFeN2NiO / Feature type: SUBJECT OF INVESTIGATION
#10: Chemical ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: Mg / Feature type: SUBJECT OF INVESTIGATION

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Details

Has ligand of interestY
Has protein modificationN

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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: Quaternary complex of the NADPH-bound Pyrococcus furiosus SHI complex
Type: COMPLEX / Entity ID: #1-#4 / Source: RECOMBINANT
Molecular weightExperimental value: NO
Source (natural)Organism: Pyrococcus furiosus (archaea)
Source (recombinant)Organism: Pyrococcus furiosus (archaea)
Buffer solutionpH: 7.5
SpecimenConc.: 2 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: GOLD / Grid mesh size: 300 divisions/in. / Grid type: Quantifoil R1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK II / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 283.15 K

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: TFS KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Calibrated magnification: 105000 X / Nominal defocus max: 1800 nm / Nominal defocus min: 1300 nm / Cs: 2.7 mm / C2 aperture diameter: 100 µm / Alignment procedure: COMA FREE
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingAverage exposure time: 1.5 sec. / Electron dose: 60 e/Å2 / Film or detector model: GATAN K3 (6k x 4k) / Num. of real images: 31397
EM imaging opticsEnergyfilter name: GIF Bioquantum / Energyfilter slit width: 20 eV

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Processing

EM software
IDNameVersionCategory
1cryoSPARCparticle selection
2SerialEM4image acquisition
4cryoSPARCCTF correction
7UCSF ChimeraXmodel fitting
9PHENIXmodel refinement
10cryoSPARCinitial Euler assignment
11cryoSPARCfinal Euler assignment
12cryoSPARCclassification
13cryoSPARC3D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 1215778
3D reconstructionResolution: 3.06 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 313849 / Symmetry type: POINT
Atomic model buildingProtocol: AB INITIO MODEL
Atomic model buildingSource name: AlphaFold / Type: in silico model
RefinementHighest resolution: 3.06 Å
Stereochemistry target values: REAL-SPACE (WEIGHTED MAP SUM AT ATOM CENTERS)
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00321915
ELECTRON MICROSCOPYf_angle_d0.49729736
ELECTRON MICROSCOPYf_dihedral_angle_d12.0338218
ELECTRON MICROSCOPYf_chiral_restr0.0433193
ELECTRON MICROSCOPYf_plane_restr0.0043752

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