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Cyanide binding to cytochrome c peroxidase (H52L)
Anil Bidwai1, Misty Witt, Miriam Foshay
1Department of Chemistry and Biochemistry, Northern Illinois University, DeKalb, Illinois 60115, USA.
Biochemistry
|September 10, 2003
Summary
This study reveals that a mutant cytochrome c peroxidase (CcP) with a histidine-to-leucine change preferentially binds cyanide anions, unlike the wild-type enzyme. This binding involves a pH-dependent isomerization, stabilizing the complex.
Area of Science:
- Biochemistry
- Enzyme kinetics
- Protein engineering
Background:
- Cytochrome c peroxidase (CcP) is crucial in cellular redox processes.
- Understanding enzyme-ligand interactions, like cyanide binding, is key to enzyme function.
- Distal residue mutations can significantly alter enzyme active site properties.
Purpose of the Study:
- To investigate cyanide binding to a CcP variant (CcP(H52L)) with a distal histidine replaced by leucine.
- To characterize the pH-dependent binding kinetics and thermodynamics of CcP(H52L) with cyanide.
- To compare cyanide binding mechanisms between CcP(H52L) and wild-type CcP.
Main Methods:
- Spectroscopic analysis
- Equilibrium binding studies
- Kinetic measurements (including pH-jump techniques)
Main Results:
- CcP(H52L) preferentially binds cyanide anions, unlike wild-type CcP which binds HCN.
- Binding affinity (dissociation constant) varies significantly with pH (60-fold change).
- Kinetics are biphasic, involving association and a pH-dependent isomerization that stabilizes the complex.
Conclusions:
- The distal histidine in CcP plays a critical role in determining the cyanide binding pathway.
- The CcP(H52L) variant exhibits unique pH-dependent binding kinetics and a novel isomerization step.
- These findings offer insights into enzyme active site plasticity and ligand interactions.