1JRY

Crystal structure of Arg402Lys mutant flavocytochrome c3 from Shewanella frigidimarina


Experimental Data Snapshot

  • Method: X-RAY DIFFRACTION
  • Resolution: 2.00 Å
  • R-Value Free: 0.236 
  • R-Value Work: 0.157 

wwPDB Validation   3D Report Full Report


This is version 2.0 of the entry. See complete history


Literature

Kinetic and crystallographic analysis of the key active site acid/base arginine in a soluble fumarate reductase.

Mowat, C.G.Moysey, R.Miles, C.S.Leys, D.Doherty, M.K.Taylor, P.Walkinshaw, M.D.Reid, G.A.Chapman, S.K.

(2001) Biochemistry 40: 12292-12298

  • DOI: https://doi.org/10.1021/bi011360h
  • Primary Citation of Related Structures:  
    1JRX, 1JRY, 1JRZ

  • PubMed Abstract: 

    There is now overwhelming evidence supporting a common mechanism for fumarate reduction in the respiratory fumarate reductases. The X-ray structures of substrate-bound forms of these enzymes indicate that the substrate is well positioned to accept a hydride from FAD and a proton from an arginine side chain. Recent work on the enzyme from Shewanella frigidimarina [Doherty, M. K., Pealing, S. L., Miles, C. S., Moysey, R., Taylor, P., Walkinshaw, M. D., Reid, G. A., and Chapman, S. K. (2000) Biochemistry 39, 10695-10701] has strengthened the assignment of an arginine (Arg402) as the proton donor in fumarate reduction. Here we describe the crystallographic and kinetic analyses of the R402A, R402K, and R402Y mutant forms of the Shewanella enzyme. The crystal structure of the R402A mutant (2.0 A resolution) shows it to be virtually identical to the wild-type enzyme, apart from the fact that a water molecule occupies the position previously taken by part of the guanidine group of R402. Although structurally similar to the wild-type enzyme, the R402A mutant is inactive under all the conditions that were studied. This implies that a water molecule, in this position in the active site, cannot function as the proton donor for fumarate reduction. In contrast to the R402A mutation, both the R402K and R402Y mutant enzymes are active. Although this activity was at a very low level (at pH 7.2 some 10(4)-fold lower than that for the wild type), it does imply that both lysine and tyrosine can fulfill the role of an active site proton donor, albeit very poorly. The crystal structures of the R402K and R402Y mutant enzymes (2.0 A resolution) show that distances from the lysine and tyrosine side chains to the nearest carbon atom of fumarate are approximately 3.5 A, clearly permitting proton transfer. The combined results from mutagenesis, crystallographic, and kinetic studies provide formidable evidence that R402 acts as both a Lewis acid (stabilizing the build-up of negative charge upon hydride transfer from FAD to fumarate) and a Brønsted acid (donating the proton to the substrate to complete the formation of succinate).


  • Organizational Affiliation

    Department of Chemistry, University of Edinburgh, West Mains Road, Edinburgh EH9 3JJ, UK.


Macromolecules
Find similar proteins by:  (by identity cutoff)  |  3D Structure
Entity ID: 1
MoleculeChains Sequence LengthOrganismDetailsImage
FLAVOCYTOCHROME C
A, B
571Shewanella frigidimarinaMutation(s): 1 
Gene Names: fcc
EC: 1.3.99.1
UniProt
Find proteins for P0C278 (Shewanella frigidimarina)
Explore P0C278 
Go to UniProtKB:  P0C278
Entity Groups  
Sequence Clusters30% Identity50% Identity70% Identity90% Identity95% Identity100% Identity
UniProt GroupP0C278
Sequence Annotations
Expand
  • Reference Sequence
Small Molecules
Ligands 4 Unique
IDChains Name / Formula / InChI Key2D Diagram3D Interactions
FAD
Query on FAD

Download Ideal Coordinates CCD File 
H [auth A],
O [auth B]
FLAVIN-ADENINE DINUCLEOTIDE
C27 H33 N9 O15 P2
VWWQXMAJTJZDQX-UYBVJOGSSA-N
HEM
Query on HEM

Download Ideal Coordinates CCD File 
D [auth A]
E [auth A]
F [auth A]
G [auth A]
K [auth B]
D [auth A],
E [auth A],
F [auth A],
G [auth A],
K [auth B],
L [auth B],
M [auth B],
N [auth B]
PROTOPORPHYRIN IX CONTAINING FE
C34 H32 Fe N4 O4
KABFMIBPWCXCRK-RGGAHWMASA-L
FUM
Query on FUM

Download Ideal Coordinates CCD File 
I [auth A],
P [auth B]
FUMARIC ACID
C4 H4 O4
VZCYOOQTPOCHFL-OWOJBTEDSA-N
NA
Query on NA

Download Ideal Coordinates CCD File 
C [auth A],
J [auth B]
SODIUM ION
Na
FKNQFGJONOIPTF-UHFFFAOYSA-N
Experimental Data & Validation

Experimental Data

  • Method: X-RAY DIFFRACTION
  • Resolution: 2.00 Å
  • R-Value Free: 0.236 
  • R-Value Work: 0.157 
  • Space Group: P 1 21 1
Unit Cell:
Length ( Å )Angle ( ˚ )
a = 76.612α = 90
b = 86.916β = 104.48
c = 88.985γ = 90
Software Package:
Software NamePurpose
AMoREphasing
REFMACrefinement
DENZOdata reduction
SCALEPACKdata scaling

Structure Validation

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Entry History 

Deposition Data

Revision History  (Full details and data files)

  • Version 1.0: 2001-11-02
    Type: Initial release
  • Version 1.1: 2008-04-27
    Changes: Version format compliance
  • Version 1.2: 2011-07-13
    Changes: Version format compliance
  • Version 1.3: 2021-10-27
    Changes: Database references, Derived calculations
  • Version 1.4: 2023-08-16
    Changes: Data collection, Refinement description
  • Version 2.0: 2023-11-15
    Changes: Atomic model, Data collection