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- PDB-28pn: CryoEM structure of native quinol dependent Nitric Oxide Reductas... -

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Basic information

Entry
Database: PDB / ID: 28pn
TitleCryoEM structure of native quinol dependent Nitric Oxide Reductase at pH 8.0 on gold grid.
ComponentsNitric oxide reductase subunit B
KeywordsMEMBRANE PROTEIN / quinol-dependent Nitric Oxide Reductase
Function / homology
Function and homology information


nitric oxide reductase (cytochrome c) / nitric oxide reductase activity
Similarity search - Function
: / Nitric oxide reductase subunit B, cytochrome c-like domain / Cytochrome c oxidase subunit I / Cytochrome c oxidase-like, subunit I superfamily / Cytochrome C and Quinol oxidase polypeptide I
Similarity search - Domain/homology
: / PROTOPORPHYRIN IX CONTAINING FE / Nitric oxide reductase subunit B
Similarity search - Component
Biological speciesAchromobacter xylosoxidans (bacteria)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.7 Å
AuthorsKhaja, F. / Antonyuk, S.V. / Muench, S.P. / Hasnain, S.S.
Funding support United Kingdom, 1items
OrganizationGrant numberCountry
Biotechnology and Biological Sciences Research Council (BBSRC)BB/X015491/1 United Kingdom
CitationJournal: ACS Bio Med Chem Au / Year: 2026
Title: CryoEM Structures of Native Quinol-Dependent Nitric Oxide Reductase in Resting and Quinol-Bound States.
Authors: Faisal T Khaja / Allegra Mboukou / Louie P Aspinall / Charlotte E Hawksworth / Robert R Eady / Svetlana V Antonyuk / Stephen P Muench / S Samar Hasnain /
Abstract: The membrane-bound quinol-dependent nitric oxide reductases (qNORs), which are members of the respiratory heme-copper oxidase superfamily, are of major importance to food production, environment, and ...The membrane-bound quinol-dependent nitric oxide reductases (qNORs), which are members of the respiratory heme-copper oxidase superfamily, are of major importance to food production, environment, and human health. They are unique to bacteria and catalyze N-N bond formation, converting nitric oxide (NO) to generate the enzymatic product, nitrous oxide (NO), in agricultural and pathogenic conditions. High-resolution qNOR structures have been reported from two bacterial species, in which the molecular size of the protein was increased by the insertion of apocytochrome b (BRIL) at the C-terminus to facilitate cryoEM structure determination. However, it remains uncertain how BRIL fusion alters the native structure of these metalloenzymes. Here, we present the first high-resolution structure of qNOR (qNOR) determined without a fusion tag at two different pH values, revealing structural differences near the catalytic core as well as overall conformational changes between the native and fusion-tagged structures. The native enzyme shows a bell-shaped pH dependence of enzymatic activity, like nitrite reductase, the preceding enzyme in the denitrification pathway, which generates the substrate NO. In addition, we report structures of qNOR bound to quinol and hydroxyquinol that provide valuable insight into the potential electron transfer pathway originating from Trp718 to the redox centers.
History
DepositionFeb 12, 2026Deposition site: PDBE / Processing site: PDBE
Revision 1.0May 6, 2026Provider: repository / Type: Initial release
Revision 1.0May 6, 2026Data content type: EM metadata / Data content type: EM metadata / Provider: repository / Type: Initial release
Revision 1.0May 6, 2026Data content type: FSC / Data content type: FSC / Provider: repository / Type: Initial release
Revision 1.0May 6, 2026Data content type: Half map / Part number: 1 / Data content type: Half map / Provider: repository / Type: Initial release
Revision 1.0May 6, 2026Data content type: Half map / Part number: 2 / Data content type: Half map / Provider: repository / Type: Initial release
Revision 1.0May 6, 2026Data content type: Image / Data content type: Image / Provider: repository / Type: Initial release
Revision 1.0May 6, 2026Data content type: Mask / Part number: 1 / Data content type: Mask / Provider: repository / Type: Initial release
Revision 1.0May 6, 2026Data content type: Primary map / Data content type: Primary map / Provider: repository / Type: Initial release

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
A: Nitric oxide reductase subunit B
B: Nitric oxide reductase subunit B
hetero molecules


Theoretical massNumber of molelcules
Total (without water)172,10810
Polymers169,4502
Non-polymers2,6588
Water9,872548
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: electron microscopy, not applicable
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1

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Components

#1: Protein Nitric oxide reductase subunit B / Nitric-oxide reductase large subunit


Mass: 84724.867 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Achromobacter xylosoxidans (bacteria) / Gene: norB_1, ERS451415_02175, IUJ48_17015 / Production host: Escherichia coli BL21(DE3) (bacteria)
References: UniProt: A0ABF7PH53, nitric oxide reductase (cytochrome c)
#2: Chemical
ChemComp-HEM / PROTOPORPHYRIN IX CONTAINING FE / HEME


Mass: 616.487 Da / Num. of mol.: 4 / Source method: obtained synthetically / Formula: C34H32FeN4O4 / Feature type: SUBJECT OF INVESTIGATION
#3: Chemical ChemComp-CA / CALCIUM ION


Mass: 40.078 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: Ca
#4: Chemical ChemComp-FE / FE (III) ION


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


Mass: 18.015 Da / Num. of mol.: 548 / Source method: isolated from a natural source / Formula: H2O
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: quinol-dependent Nitric Oxide Reductase at pH 8.0 on gold grid.
Type: COMPLEX
Details: Two HEM and one Fe are the cofactors covalently bound to this protein. Fe is linked with one of the HEM by a water molecule via mu oxo bridge.
Entity ID: #1 / Source: RECOMBINANT
Source (natural)Organism: Achromobacter xylosoxidans (bacteria)
Source (recombinant)Organism: Escherichia coli BL21(DE3) (bacteria)
Buffer solutionpH: 8
SpecimenConc.: 5 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: GOLD
VitrificationCryogen name: ETHANE

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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 / Nominal defocus max: 2700 nm / Nominal defocus min: 700 nm
Image recordingElectron dose: 40 e/Å2 / Film or detector model: FEI FALCON I (4k x 4k)

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Processing

EM software
IDNameVersionCategory
1cryoSPARCparticle selection
2PHENIX1.21.1_5286model refinement
13cryoSPARC3D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
3D reconstructionResolution: 2.7 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 91995 / Symmetry type: POINT
RefinementHighest resolution: 2.7 Å
Stereochemistry target values: REAL-SPACE (WEIGHTED MAP SUM AT ATOM CENTERS)
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00411676
ELECTRON MICROSCOPYf_angle_d0.68215993
ELECTRON MICROSCOPYf_dihedral_angle_d6.7631555
ELECTRON MICROSCOPYf_chiral_restr0.0411677
ELECTRON MICROSCOPYf_plane_restr0.0051991

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