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Tyr-143 facilitates interdomain electron transfer in flavocytochrome b2
C S Miles1, N Rouvière-Fourmy, F Lederer
1Edinburgh Centre for Molecular Recognition, Department of Chemistry, University of Edinburgh, Scotland, U.K.
The Biochemical Journal
|July 1, 1992
Summary
Replacing Tyr-143 with phenylalanine in flavocytochrome b2 significantly alters electron transfer. The mutation shifts the rate-determining step from lactate oxidation to interdomain electron transfer between FMN and heme groups.
Area of Science:
- Biochemistry
- Enzyme kinetics
- Protein engineering
Background:
- Flavocytochrome b2 (L-lactate:cytochrome c oxidoreductase) is crucial for electron transfer.
- Understanding the catalytic cycle and the role of specific residues is key to enzyme function.
Purpose of the Study:
- To investigate the role of Tyr-143 in the catalytic cycle of flavocytochrome b2.
- To examine the impact of mutating Tyr-143 to phenylalanine on electron transfer steps.
Main Methods:
- Utilized steady-state and stopped-flow kinetic methods.
- Employed kinetic-isotope effect studies with [2-2H]lactate.
- Investigated electron transfer using external acceptors like cytochrome c and ferricyanide.
Main Results:
- The Tyr-143 to Phenylalanine mutation shifted the rate-determining step from proton abstraction to interdomain electron transfer.
- Haem reduction rate decreased over 20-fold in the mutant enzyme.
- Kinetic-isotope effects for both flavin and haem reduction were significantly reduced in the mutant.
Conclusions:
- Tyr-143 plays a critical role in facilitating interdomain electron transfer between the FMN and haem prosthetic groups in flavocytochrome b2.
- The mutation dramatically affects the enzyme's catalytic efficiency by altering the rate-limiting step.