1LJ1

Crystal structure of Q363F/R402A mutant flavocytochrome c3


Experimental Data Snapshot

  • Method: X-RAY DIFFRACTION
  • Resolution: 2.00 Å
  • R-Value Free: 0.235 
  • R-Value Work: 0.161 

wwPDB Validation   3D Report Full Report


This is version 2.0 of the entry. See complete history


Literature

Engineering Water to act as the active site acid catalyst in a soluble fumarate reductase

Mowat, C.G.Pankhurst, K.L.Miles, C.S.Leys, D.Walkinshaw, M.D.Reid, G.A.Chapman, S.K.

(2002) Biochemistry 41: 11990-11996

  • DOI: https://doi.org/10.1021/bi0203177
  • Primary Citation of Related Structures:  
    1LJ1, 1M64

  • PubMed Abstract: 

    The ability of an arginine residue to function as the active site acid catalyst in the fumarate reductase family of enzymes is now well-established. Recently, a dual role for the arginine during fumarate reduction has been proposed [Mowat, C. G., Moysey, R., Miles, C. S., Leys, D., Doherty, M. K., Taylor, P., Walkinshaw, M. D., Reid, G. A., and Chapman, S. K. (2001) Biochemistry 40, 12292-12298] in which it acts both as a Lewis acid in transition-state stabilization and as a Brønsted acid in proton delivery. This proposal has led to the prediction that, if appropriately positioned, a water molecule would be capable of functioning as the active site Brønsted acid. In this paper, we describe the construction and kinetic and crystallographic analysis of the Q363F single mutant and Q363F/R402A double mutant forms of flavocytochrome c(3), the soluble fumarate reductase from Shewanella frigidimarina. Although replacement of the active site acid, Arg402, with alanine has been shown to eliminate fumarate reductase activity, this phenomenon is partially reversed by the additional substitution of Gln363 with phenylalanine. This Gln --> Phe substitution in the inactive R402A mutant enzyme was designed to "push" a water molecule close enough to the substrate C3 atom to allow it to act as a Brønsted acid. The 2.0 A resolution crystal structure of the Q363F/R402A mutant enzyme does indeed reveal the introduction of a water molecule at the correct position in the active site to allow it to act as the catalytic proton donor. The 1.8 A resolution crystal structure of the Q363F mutant enzyme shows a water molecule similarly positioned, which can account for its measured fumarate reductase activity. However, in this mutant enzyme Michaelis complex formation is impaired due to significant and unpredicted structural changes at the active site.


  • Organizational Affiliation

    Department of Chemistry, University of Edinburgh, West Mains Road, Edinburgh EH9 3JJ, Scotland, U.K.


Macromolecules
Find similar proteins by:  (by identity cutoff)  |  3D Structure
Entity ID: 1
MoleculeChains Sequence LengthOrganismDetailsImage
flavocytochrome c3
A, B
571Shewanella frigidimarinaMutation(s): 2 
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
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  • 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: 0.235 
  • R-Value Work: 0.161 
  • Space Group: P 1 21 1
Unit Cell:
Length ( Å )Angle ( ˚ )
a = 77.971α = 90
b = 88.28β = 103.89
c = 90.087γ = 90
Software Package:
Software NamePurpose
DENZOdata reduction
SCALEPACKdata scaling
AMoREphasing
REFMACrefinement

Structure Validation

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Entry History 

Deposition Data

Revision History  (Full details and data files)

  • Version 1.0: 2002-11-06
    Type: Initial release
  • Version 1.1: 2008-04-28
    Changes: Version format compliance
  • Version 1.2: 2011-07-13
    Changes: Version format compliance
  • Version 1.3: 2021-10-27
    Changes: Data collection, 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