Radical formation in cytochrome c oxidase.

Area of Science:

• Biochemistry and Biophysics
• Enzymology
• Electron Paramagnetic Resonance Spectroscopy

Background:

• The controversial role of radical formation in bovine cytochrome c oxidase (bCcO) during oxygen reduction and proton translocation remains unresolved.
• Understanding radical intermediates is crucial for elucidating the enzyme's catalytic mechanism and proton pumping activity.

Purpose of the Study:

• To investigate the formation of radicals in bCcO during O(2) redox chemistry under single turnover conditions.
• To identify the species of radicals formed and their localization (protein-based vs. reductant-based).
• To explore the potential role of specific tyrosine residues in proton translocation.

Main Methods:

• Utilized a custom-built rapid freeze quenching (RFQ) device to trap reaction intermediates at cryogenic temperatures (77K).
• Employed X-band (9GHz) and D-band (130GHz) continuous wave (CW) and pulsed electron paramagnetic resonance (EPR) spectroscopy.
• Analyzed reactions of O(2) with reduced bCcO (using ascorbate or dithionite) and H(2)O(2) with oxidized bCcO.

Main Results:

• No evidence of protein-based radicals was detected when O(2) reacted with reduced bCcO.
• Identified ascorbyl radical when using ascorbate and SO(2)(-) ion radical when using dithionite as reductants.
• Observed peroxyl (ROO) species and identified radicals on Tyr-244 and Tyr-129 in reactions with H(2)O(2), suggesting Tyr-129's role in proton transfer.

Conclusions:

• Radicals formed during the reaction of O(2) with reduced bCcO are primarily derived from the reductants, not the protein.
• Tyrosine residues, particularly Tyr-129, are implicated in the proton translocation mechanism, potentially acting as proton loading sites.
• The findings provide new insights into the redox chemistry and proton pumping mechanism of cytochrome c oxidase.