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- PDB-8a6t: Cryo-EM structure of the electron bifurcating Fe-Fe hydrogenase H... -

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Basic information

Entry
Database: PDB / ID: 8a6t
TitleCryo-EM structure of the electron bifurcating Fe-Fe hydrogenase HydABC complex from Thermoanaerobacter kivui in the reduced state
Components(Electron bifurcating hydrogenase subunit ...) x 3
KeywordsELECTRON TRANSPORT / flavin-based electron bifurcating hydrogenase / Fe-Fe hydrogenase / NAD(p) / FMN
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:ubiquinone oxidoreductase, subunit G, iron-sulphur binding / His(Cys)3-ligated-type [4Fe-4S] domain profile. / NADH-quinone oxidoreductase subunit E-like / NADH-quinone oxidoreductase subunit E, N-terminal / 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 / 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
FE2/S2 (INORGANIC) CLUSTER / FLAVIN MONONUCLEOTIDE / Chem-HC1 / NADP NICOTINAMIDE-ADENINE-DINUCLEOTIDE PHOSPHATE / IRON/SULFUR CLUSTER / Electron bifurcating hydrogenase subunit HydA1 / Electron bifurcating hydrogenase subunit HydB / Electron bifurcating hydrogenase subunit HydC
Similarity search - Component
Biological speciesThermoanaerobacter kivui (bacteria)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.1 Å
AuthorsKumar, A. / Saura, P. / Gamiz-Hernandez, A.P. / Kaila, V.R.I. / Mueller, V. / Schuller, J.M.
Funding support Germany, European Union, Sweden, 6items
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, 2022Deposition site: PDBE / Processing site: PDBE
Revision 1.0Feb 15, 2023Provider: repository / Type: Initial release
Revision 1.1Mar 8, 2023Group: Database references / Category: citation / citation_author
Item: _citation.pdbx_database_id_PubMed / _citation.title ..._citation.pdbx_database_id_PubMed / _citation.title / _citation_author.identifier_ORCID / _citation_author.name
Revision 1.2Mar 29, 2023Group: Database references / Category: citation
Item: _citation.journal_volume / _citation.page_first / _citation.page_last

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
A: Electron bifurcating hydrogenase subunit HydA1
B: Electron bifurcating hydrogenase subunit HydB
C: Electron bifurcating hydrogenase subunit HydC
D: Electron bifurcating hydrogenase subunit HydA1
E: Electron bifurcating hydrogenase subunit HydB
F: Electron bifurcating hydrogenase subunit HydC
hetero molecules


Theoretical massNumber of molelcules
Total (without water)310,80934
Polymers301,5896
Non-polymers9,22028
Water00
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: electron microscopy
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area30420 Å2
ΔGint-528 kcal/mol
Surface area106070 Å2
MethodPISA

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Components

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Electron bifurcating hydrogenase subunit ... , 3 types, 6 molecules ADBECF

#1: Protein Electron bifurcating hydrogenase subunit HydA1


Mass: 63890.727 Da / Num. of mol.: 2 / Source method: isolated from a natural source / Source: (natural) Thermoanaerobacter kivui (bacteria)
References: UniProt: A0A097ATG3, hydrogen dehydrogenase (NADP+)
#2: Protein Electron bifurcating hydrogenase subunit HydB


Mass: 68279.047 Da / Num. of mol.: 2 / Source method: isolated from a natural source / Source: (natural) Thermoanaerobacter kivui (bacteria)
References: UniProt: A0A097ATG4, hydrogen dehydrogenase (NADP+)
#3: Protein Electron bifurcating hydrogenase subunit HydC


Mass: 18624.678 Da / Num. of mol.: 2 / Source method: isolated from a natural source / Source: (natural) Thermoanaerobacter kivui (bacteria)
References: UniProt: A0A097ATI0, hydrogen dehydrogenase (NADP+)

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Non-polymers , 6 types, 28 molecules

#4: 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
#5: Chemical ChemComp-HC1 / 2 IRON/2 SULFUR/5 CARBONYL/2 WATER INORGANIC CLUSTER


Mass: 355.933 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: C5H8Fe2O7S2 / Feature type: SUBJECT OF INVESTIGATION
#6: Chemical
ChemComp-FES / FE2/S2 (INORGANIC) CLUSTER


Mass: 175.820 Da / Num. of mol.: 6 / Source method: obtained synthetically / Formula: Fe2S2 / Feature type: SUBJECT OF INVESTIGATION
#7: Chemical ChemComp-FMN / FLAVIN MONONUCLEOTIDE / RIBOFLAVIN MONOPHOSPHATE


Mass: 456.344 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: C17H21N4O9P / Feature type: SUBJECT OF INVESTIGATION
#8: Chemical ChemComp-ZN / ZINC ION


Mass: 65.409 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: Zn / Feature type: SUBJECT OF INVESTIGATION
#9: 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

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Details

Has ligand of interestY

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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: Electron bifurcating Fe-Fe hydrogenase HydABC complex from Thermoanaerobacter kivui
Type: COMPLEX / Entity ID: #1-#3 / Source: NATURAL
Molecular weightValue: 0.348 MDa / Experimental value: YES
Source (natural)Organism: Thermoanaerobacter kivui (bacteria)
Buffer solutionpH: 7.6
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
VitrificationCryogen name: ETHANE-PROPANE

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Electron microscopy imaging

MicroscopyModel: JEOL CRYO ARM 200
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 200 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: GATAN K2 SUMMIT (4k x 4k)

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Processing

SoftwareName: PHENIX / Version: 1.19.2_4158: / Classification: refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
3D reconstructionResolution: 3.1 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 193719 / Symmetry type: POINT
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00221558
ELECTRON MICROSCOPYf_angle_d0.4829156
ELECTRON MICROSCOPYf_dihedral_angle_d5.0732952
ELECTRON MICROSCOPYf_chiral_restr0.0423218
ELECTRON MICROSCOPYf_plane_restr0.0043700

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