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- PDB-8enm: CryoEM structure of the high pH nitrogenase MoFe-protein under no... -

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Entry
Database: PDB / ID: 8enm
TitleCryoEM structure of the high pH nitrogenase MoFe-protein under non-turnover conditions
Components(Nitrogenase molybdenum-iron protein ...) x 2
KeywordsOXIDOREDUCTASE / nitrogenase / nitrogen fixation / reductase / MoFe
Function / homology
Function and homology information


molybdenum-iron nitrogenase complex / nitrogenase / nitrogenase activity / nitrogen fixation / iron-sulfur cluster binding / ATP binding / metal ion binding
Similarity search - Function
Nitrogenase molybdenum-iron protein beta chain, N-terminal / Domain of unknown function (DUF3364) / Nitrogenase molybdenum-iron protein alpha chain / Nitrogenase molybdenum-iron protein beta chain / Nitrogenase component 1, alpha chain / Nitrogenase component 1, conserved site / Nitrogenases component 1 alpha and beta subunits signature 2. / Nitrogenases component 1 alpha and beta subunits signature 1. / Nitrogenase/oxidoreductase, component 1 / : / Nitrogenase component 1 type Oxidoreductase
Similarity search - Domain/homology
FE(8)-S(7) CLUSTER / : / 3-HYDROXY-3-CARBOXY-ADIPIC ACID / Chem-ICS / Nitrogenase molybdenum-iron protein alpha chain / Nitrogenase molybdenum-iron protein beta chain
Similarity search - Component
Biological speciesAzotobacter vinelandii (bacteria)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.14 Å
AuthorsWarmack, R.A. / Maggiolo, A.O. / Rees, D.C.
Funding support United States, 3items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)GM045162 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)GM143836-01 United States
Howard Hughes Medical Institute (HHMI) United States
CitationJournal: Nat Commun / Year: 2023
Title: Structural consequences of turnover-induced homocitrate loss in nitrogenase.
Authors: Rebeccah A Warmack / Ailiena O Maggiolo / Andres Orta / Belinda B Wenke / James B Howard / Douglas C Rees /
Abstract: Nitrogenase catalyzes the ATP-dependent reduction of dinitrogen to ammonia during the process of biological nitrogen fixation that is essential for sustaining life. The active site FeMo-cofactor ...Nitrogenase catalyzes the ATP-dependent reduction of dinitrogen to ammonia during the process of biological nitrogen fixation that is essential for sustaining life. The active site FeMo-cofactor contains a [7Fe:1Mo:9S:1C] metallocluster coordinated with an R-homocitrate (HCA) molecule. Here, we establish through single particle cryoEM and chemical analysis of two forms of the Azotobacter vinelandii MoFe-protein - a high pH turnover inactivated species and a ∆NifV variant that cannot synthesize HCA - that loss of HCA is coupled to α-subunit domain and FeMo-cofactor disordering, and formation of a histidine coordination site. We further find a population of the ∆NifV variant complexed to an endogenous protein identified through structural and proteomic approaches as the uncharacterized protein NafT. Recognition by endogenous NafT demonstrates the physiological relevance of the HCA-compromised form, perhaps for cofactor insertion or repair. Our results point towards a dynamic active site in which HCA plays a role in enabling nitrogenase catalysis by facilitating activation of the FeMo-cofactor from a relatively stable form to a state capable of reducing dinitrogen under ambient conditions.
History
DepositionSep 30, 2022Deposition site: RCSB / Processing site: RCSB
Revision 1.0Mar 8, 2023Provider: repository / Type: Initial release
Revision 1.1Jun 19, 2024Group: Data collection / Category: chem_comp_atom / chem_comp_bond

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Structure visualization

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MolmilJmol/JSmol

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Assembly

Deposited unit
A: Nitrogenase molybdenum-iron protein alpha chain
B: Nitrogenase molybdenum-iron protein beta chain
C: Nitrogenase molybdenum-iron protein alpha chain
D: Nitrogenase molybdenum-iron protein beta chain
hetero molecules


Theoretical massNumber of molelcules
Total (without water)233,23912
Polymers229,7984
Non-polymers3,4418
Water30,4991693
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: electron microscopy
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

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Nitrogenase molybdenum-iron protein ... , 2 types, 4 molecules ACBD

#1: Protein Nitrogenase molybdenum-iron protein alpha chain / Dinitrogenase / Nitrogenase component I


Mass: 55363.043 Da / Num. of mol.: 2 / Source method: isolated from a natural source / Source: (natural) Azotobacter vinelandii (bacteria) / References: UniProt: P07328, nitrogenase
#2: Protein Nitrogenase molybdenum-iron protein beta chain / Dinitrogenase / Nitrogenase component I


Mass: 59535.879 Da / Num. of mol.: 2 / Source method: isolated from a natural source / Source: (natural) Azotobacter vinelandii (bacteria) / References: UniProt: P07329, nitrogenase

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Non-polymers , 5 types, 1701 molecules

#3: Chemical ChemComp-ICS / iron-sulfur-molybdenum cluster with interstitial carbon


Mass: 787.451 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: CFe7MoS9
#4: Chemical ChemComp-HCA / 3-HYDROXY-3-CARBOXY-ADIPIC ACID


Mass: 206.150 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: C7H10O7
#5: Chemical ChemComp-CLF / FE(8)-S(7) CLUSTER


Mass: 671.215 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: Fe8S7
#6: Chemical ChemComp-FE / FE (III) ION


Mass: 55.845 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: Fe
#7: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 1693 / Source method: isolated from a natural source / Formula: H2O

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Details

Has ligand of interestN

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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: Heterotetrameric nitrogenase MoFe-protein / Type: COMPLEX / Entity ID: #1-#2 / Source: NATURAL
Source (natural)Organism: Azotobacter vinelandii (bacteria)
Buffer solutionpH: 7.8
SpecimenConc.: 4.75 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
VitrificationCryogen name: ETHANE-PROPANE

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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: OTHER
Electron lensMode: BRIGHT FIELD / Nominal defocus max: -3000 nm / Nominal defocus min: -800 nm
Image recordingElectron dose: 60 e/Å2 / Film or detector model: GATAN K3 (6k x 4k)

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Processing

SoftwareName: PHENIX / Version: 1.20.1_4487: / Classification: refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
3D reconstructionResolution: 2.14 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 331604 / Symmetry type: POINT
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00316454
ELECTRON MICROSCOPYf_angle_d0.52522344
ELECTRON MICROSCOPYf_dihedral_angle_d5.3292278
ELECTRON MICROSCOPYf_chiral_restr0.0442355
ELECTRON MICROSCOPYf_plane_restr0.0042856

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