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Electron Transport Chain: Complex III and IV01:43

Electron Transport Chain: Complex III and IV

During the electron transport chain, electrons from NADH and FADH2 are first transferred to complexes I and II, respectively. These two complexes then transfer the electrons to ubiquinol, which carries them further to complex III. Complex III passes the electrons across the intermembrane space to Cyt c, which carries them further to complex IV. Complex IV donates electrons to oxygen and reduces it to water. As electrons pass through complexes I, III, and IV, the energy released aids the pumping...

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A Rapid and Specific Microplate Assay for the Determination of Intra- and Extracellular Ascorbate in Cultured Cells
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Engineering ascorbate peroxidase activity into cytochrome c peroxidase.

Yergalem T Meharenna1, Patricia Oertel, B Bhaskar

  • 1Department of Molecular Biology and Biochemistry, University of California, Irvine, California 92697-3900, USA.

Biochemistry
|September 6, 2008
PubMed
Summary
This summary is machine-generated.

Engineered cytochrome c peroxidase (CCP) gained ascorbate peroxidase (APX) activity by altering its substrate binding site. This modification demonstrates how structural changes can confer novel enzymatic functions, highlighting protein adaptability.

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Area of Science:

  • Biochemistry
  • Structural Biology
  • Enzyme Engineering

Background:

  • Cytochrome c peroxidase (CCP) and ascorbate peroxidase (APX) share similar structures but lack cross-activity.
  • APX possesses a unique ascorbate-binding site involving heme propionates and a surface Arginine (Arg).
  • CCP has a distinct surface loop and an Asparagine (Asn) residue in the corresponding region, preventing ascorbate binding.

Purpose of the Study:

  • To engineer CCP to exhibit APX activity by modifying its substrate-binding site.
  • To investigate the structural basis for the substrate specificity differences between CCP and APX.

Main Methods:

  • Protein engineering of CCP to create the CCP2APX mutant by introducing the APX ascorbate-binding loop and critical Arg residue.
  • X-ray crystallography to determine the structure of the CCP2APX mutant.
  • Enzymatic assays to measure the activity of the mutant enzyme.

Main Results:

  • The crystal structure of the CCP2APX mutant revealed an engineered site nearly identical to that of APX.
  • Wild-type CCP exhibited no APX activity.
  • The CCP2APX mutant catalyzed the peroxidation of ascorbate at a significant rate (approximately 12 min⁻¹).

Conclusions:

  • The engineered ascorbate-binding loop in CCP2APX is capable of binding ascorbate.
  • Structural modifications can successfully confer novel substrate specificity and activity to enzymes.
  • This study provides insights into the structure-function relationships of peroxidases.