3OJW

Disulfide crosslinked cytochrome P450 reductase inactive


Experimental Data Snapshot

  • Method: X-RAY DIFFRACTION
  • Resolution: 2.20 Å
  • R-Value Free: 0.251 
  • R-Value Work: 0.205 
  • R-Value Observed: 0.205 

Starting Model: experimental
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Ligand Structure Quality Assessment 


This is version 1.3 of the entry. See complete history


Literature

Conformational Changes of NADPH-Cytochrome P450 Oxidoreductase Are Essential for Catalysis and Cofactor Binding.

Xia, C.Hamdane, D.Shen, A.L.Choi, V.Kasper, C.B.Pearl, N.M.Zhang, H.Im, S.C.Waskell, L.Kim, J.J.

(2011) J Biol Chem 286: 16246-16260

  • DOI: https://doi.org/10.1074/jbc.M111.230532
  • Primary Citation of Related Structures:  
    3OJW, 3OJX

  • PubMed Abstract: 

    The crystal structure of NADPH-cytochrome P450 reductase (CYPOR) implies that a large domain movement is essential for electron transfer from NADPH via FAD and FMN to its redox partners. To test this hypothesis, a disulfide bond was engineered between residues Asp(147) and Arg(514) in the FMN and FAD domains, respectively. The cross-linked form of this mutant protein, designated 147CC514, exhibited a significant decrease in the rate of interflavin electron transfer and large (≥90%) decreases in rates of electron transfer to its redox partners, cytochrome c and cytochrome P450 2B4. Reduction of the disulfide bond restored the ability of the mutant to reduce its redox partners, demonstrating that a conformational change is essential for CYPOR function. The crystal structures of the mutant without and with NADP(+) revealed that the two flavin domains are joined by a disulfide linkage and that the relative orientations of the two flavin rings are twisted ∼20° compared with the wild type, decreasing the surface contact area between the two flavin rings. Comparison of the structures without and with NADP(+) shows movement of the Gly(631)-Asn(635) loop. In the NADP(+)-free structure, the loop adopts a conformation that sterically hinders NADP(H) binding. The structure with NADP(+) shows movement of the Gly(631)-Asn(635) loop to a position that permits NADP(H) binding. Furthermore, comparison of these mutant and wild type structures strongly suggests that the Gly(631)-Asn(635) loop movement controls NADPH binding and NADP(+) release; this loop movement in turn facilitates the flavin domain movement, allowing electron transfer from FMN to the CYPOR redox partners.


  • Organizational Affiliation

    Department of Biochemistry, Medical College of Wisconsin, Milwaukee, Wisconsin 53226, USA.


Macromolecules
Find similar proteins by:  (by identity cutoff)  |  3D Structure
Entity ID: 1
MoleculeChains Sequence LengthOrganismDetailsImage
NADPH-cytochrome P450 reductase622Rattus norvegicusMutation(s): 8 
Gene Names: CYPORPor
EC: 1.6.2.4
UniProt
Find proteins for P00388 (Rattus norvegicus)
Explore P00388 
Go to UniProtKB:  P00388
Entity Groups  
Sequence Clusters30% Identity50% Identity70% Identity90% Identity95% Identity100% Identity
UniProt GroupP00388
Sequence Annotations
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  • Reference Sequence
Small Molecules
Experimental Data & Validation

Experimental Data

  • Method: X-RAY DIFFRACTION
  • Resolution: 2.20 Å
  • R-Value Free: 0.251 
  • R-Value Work: 0.205 
  • R-Value Observed: 0.205 
  • Space Group: P 21 21 21
Unit Cell:
Length ( Å )Angle ( ˚ )
a = 65.803α = 90
b = 72.958β = 90
c = 138.208γ = 90
Software Package:
Software NamePurpose
HKL-2000data collection
PHASERphasing
CNSrefinement
HKL-2000data reduction
HKL-2000data scaling

Structure Validation

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Ligand Structure Quality Assessment 


Entry History 

Deposition Data

Revision History  (Full details and data files)

  • Version 1.0: 2011-02-23
    Type: Initial release
  • Version 1.1: 2011-07-13
    Changes: Version format compliance
  • Version 1.2: 2018-03-07
    Changes: Data collection
  • Version 1.3: 2023-09-06
    Changes: Data collection, Database references, Derived calculations, Refinement description