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Probing domain mobility in a flavocytochrome
Emma L Rothery1, Christopher G Mowat, Caroline S Miles
1School of Chemistry, University of Edinburgh, West Mains Road, Edinburgh, EH9 3JJ, United Kingdom.
Biochemistry
|April 28, 2004
Summary
Restricting clamp domain mobility in flavocytochrome c(3) fumarate reductase via a disulfide bond slightly decreased enzyme activity. This suggests clamp domain mobility is not essential for substrate access or product release during fumarate reduction.
Area of Science:
- Biochemistry
- Structural Biology
- Enzyme Kinetics
Background:
- Fumarate reductases and succinate dehydrogenases feature a mobile clamp domain.
- This domain is hypothesized to regulate active site substrate accessibility.
Purpose of the Study:
- Investigate the role of the clamp domain's mobility in enzyme regulation.
- Determine if clamp domain mobility is essential for substrate access and product release.
Main Methods:
- Constructed a double mutant (A251C:S430C) of flavocytochrome c(3) fumarate reductase to form an interdomain disulfide bond.
- Performed crystallographic analysis (1.6 Å resolution) and kinetic studies (including solvent kinetic isotope effects).
- Confirmed disulfide bond presence in solution using Ellman analysis.
Main Results:
- A disulfide bond was successfully introduced between the FAD-binding and clamp domains, restricting their relative mobility.
- The disulfide-bridged enzyme showed a slight decrease in fumarate reduction rate compared to the non-bridged state.
- Solvent kinetic isotope studies indicated proton and/or hydride transfer as the rate-limiting step in catalysis for both wild-type and mutant enzymes.
Conclusions:
- Clamp domain mobility is not essential for regulating substrate access or product release in this enzyme.
- The catalytic mechanism is primarily limited by proton/hydride transfer, not substrate access influenced by clamp domain movement.