1Q9I: The A251c:s430c Double Mutant Of Flavocytochrome C3 From Shewanella Frigidimarina

Citation:
Abstract
The crystal structures of various different members of the family of fumarate reductases and succinate dehydrogenases have allowed the identification of a mobile clamp (or capping) domain [e.g., Taylor, P., Pealing, S. L., Reid, G. A., Chapman, S. K., and Walkinshaw, M. D. (1999) Nat. Struct. Biol. 6, 1108-1112], which has been proposed to be involved in regulating accessibility of the active site to substrate. To investigate this, we have constructed the A251C:S430C double mutant form of the soluble flavocytochrome c(3) fumarate reductase from Shewanella frigidimarina, to introduce an interdomain disulfide bond between the FAD-binding and clamp domains of the enzyme, thus restricting relative mobility between the two. Here, we describe the kinetic and crystallographic analysis of this double mutant enzyme. The 1.6 A resolution crystal structure of the A251C:S430C enzyme under oxidizing conditions reveals the formation of a disulfide bond, while Ellman analysis confirms its presence in the enzyme in solution. Kinetic analyses with the enzyme in both the nonbridged (free thiol) and the disulfide-bridged states indicate a slight decrease in the rate of fumarate reduction when the disulfide bridge is present, while solvent-kinetic-isotope studies indicate that in both wild-type and mutant enzymes the reaction is rate limited by proton and/or hydride transfer during catalysis. The limited effects of the inhibition of clamp domain mobility upon the catalytic reaction would indicate that such mobility is not essential for the regulation of substrate access or product release.
PDB ID: 1Q9IDownload
MMDB ID: 27290
PDB Deposition Date: 2003/8/25
Updated in MMDB: 2016/12
Experimental Method:
x-ray diffraction
Resolution: 1.6  Å
Source Organism:
Similar Structures:
Biological Unit for 1Q9I: monomeric; determined by author
Molecular Components in 1Q9I
Label Count Molecule
Protein (1 molecule)
1
Flavocytochrome C3
Molecule annotation
Chemicals (7 molecules)
1
1
2
4
3
1
4
1
* Click molecule labels to explore molecular sequence information.

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