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- PDB-8a5e: Cryo-EM structure of the electron bifurcating Fe-Fe hydrogenase H... -

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Entry
Database: PDB / ID: 8a5e
TitleCryo-EM structure of the electron bifurcating Fe-Fe hydrogenase HydABC complex from Acetobacterium woodii in the reduced state
Components(Iron hydrogenase ...) x 3
KeywordsELECTRON TRANSPORT / flavin-based electron bifurcating hydrogenase / Fe-Fe hydrogenase / NAD / FMN
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 / metal ion binding / membrane
Similarity search - Function
: / Iron hydrogenase, small subunit superfamily / Iron hydrogenase, subset / 4Fe-4S dicluster domain / Iron hydrogenase, small subunit / : / Iron hydrogenase small subunit / Iron hydrogenase small subunit / Iron hydrogenase, large subunit, C-terminal / Iron hydrogenase ...: / Iron hydrogenase, small subunit superfamily / Iron hydrogenase, subset / 4Fe-4S dicluster domain / 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 / NADH-quinone oxidoreductase subunit E / Soluble ligand binding domain / SLBB domain / Alpha-Beta Plaits - #20 / : / 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 / Thioredoxin-like [2Fe-2S] ferredoxin / NADH-ubiquinone oxidoreductase 51kDa subunit, FMN-binding domain / NADH-ubiquinone oxidoreductase 51kDa subunit, FMN-binding domain superfamily / Nuo51 FMN-binding domain / 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 / Glutaredoxin / Glutaredoxin / Prokaryotic membrane lipoprotein lipid attachment site profile. / Arc Repressor Mutant, subunit A / Thioredoxin-like superfamily / Alpha-Beta Plaits / 2-Layer Sandwich / Orthogonal Bundle / 3-Layer(aba) Sandwich / Mainly Alpha / Alpha Beta
Similarity search - Domain/homology
FE2/S2 (INORGANIC) CLUSTER / FLAVIN MONONUCLEOTIDE / Chem-HC1 / 1,4-DIHYDRONICOTINAMIDE ADENINE DINUCLEOTIDE / IRON/SULFUR CLUSTER / Iron hydrogenase HydA1 / Iron hydrogenase HydB
Similarity search - Component
Biological speciesAcetobacterium woodii DSM 1030 (bacteria)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.4 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 14, 2022Deposition site: PDBE / Processing site: PDBE
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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
A: Iron hydrogenase HydA1
B: Iron hydrogenase HydB
C: Iron hydrogenase HydC
D: Iron hydrogenase HydA1
hetero molecules


Theoretical massNumber of molelcules
Total (without water)215,20623
Polymers208,9114
Non-polymers6,29519
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: electron microscopy
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area18290 Å2
ΔGint-345 kcal/mol
Surface area72270 Å2

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Components

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Iron hydrogenase ... , 3 types, 4 molecules ADBC

#1: Protein Iron hydrogenase HydA1


Mass: 63692.887 Da / Num. of mol.: 2 / Source method: isolated from a natural source / Source: (natural) Acetobacterium woodii DSM 1030 (bacteria) / References: UniProt: H6LFG3, ferredoxin hydrogenase
#2: Protein Iron hydrogenase HydB


Mass: 64525.965 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Acetobacterium woodii DSM 1030 (bacteria) / References: UniProt: H6LFG4, ferredoxin hydrogenase
#3: Protein Iron hydrogenase HydC


Mass: 16999.689 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Acetobacterium woodii DSM 1030 (bacteria) / References: ferredoxin hydrogenase

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Non-polymers , 6 types, 19 molecules

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


Mass: 351.640 Da / Num. of mol.: 11 / Source method: obtained synthetically / Formula: Fe4S4 / Feature type: SUBJECT OF INVESTIGATION
#5: Chemical ChemComp-FES / FE2/S2 (INORGANIC) CLUSTER


Mass: 175.820 Da / Num. of mol.: 3 / Source method: obtained synthetically / Formula: Fe2S2 / Feature type: SUBJECT OF INVESTIGATION
#6: 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
#7: Chemical ChemComp-ZN / ZINC ION


Mass: 65.409 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: Zn / Feature type: SUBJECT OF INVESTIGATION
#8: Chemical ChemComp-FMN / FLAVIN MONONUCLEOTIDE / RIBOFLAVIN MONOPHOSPHATE


Mass: 456.344 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C17H21N4O9P / Feature type: SUBJECT OF INVESTIGATION
#9: Chemical ChemComp-NAI / 1,4-DIHYDRONICOTINAMIDE ADENINE DINUCLEOTIDE / NADH


Mass: 665.441 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C21H29N7O14P2 / Feature type: SUBJECT OF INVESTIGATION

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Details

Has ligand of interestY
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: 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

CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
3D reconstructionResolution: 3.4 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 172189 / Symmetry type: POINT

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