Functionally Distinct Bacterial Cytochrome c Peroxidases Proceed through a Common (Electro)catalytic Intermediate

Area of Science:

• Biochemistry and enzymology
• Bioinorganic chemistry
• Protein electrochemistry

Background:

• Diheme cytochrome c peroxidases (bCcPs) are crucial enzymes involved in peroxide metabolism.
• The bacterial bCcP from Shewanella oneidensis (So CcP) requires reductive activation to become catalytically competent.
• Understanding the mechanism of So CcP is essential for elucidating peroxide detoxification pathways.

Purpose of the Study:

• To investigate the mechanism of hydrogen peroxide turnover by the reductively activated So CcP.
• To identify and characterize on-pathway catalytic intermediates in So CcP.
• To compare the catalytic mechanism of So CcP with constitutively active bCcPs like Ne CcP.

Main Methods:

• Protein film voltammetry was employed to study So CcP electrochemistry and catalysis.
• Enzyme activation was achieved using sodium l-ascorbate.
• Site-directed mutagenesis was used to probe the role of active site residues and surface loops.

Main Results:

• A highly active catalytic intermediate was observed for So CcP in a high potential regime.
• The rate-limiting step involves a proton-coupled single electron reduction of a high valent iron species.
• Mutational analysis confirmed the involvement of a surface loop (loop 1) and His81 in the reductive activation process.

Conclusions:

• This study provides the first observation of an on-pathway catalytic intermediate for a reductively activated bacterial bCcP.
• Unexpected mechanistic similarities exist between reductively activated (So CcP) and constitutively active (Ne CcP) bCcPs.
• The findings highlight conserved features in the rate-limiting steps across different classes of bCcPs.