Perfusion-induced redox differences in cytochrome c oxidase: ATR/FT-IR spectroscopy

Area of Science:

• Biochemistry
• Spectroscopy

Background:

• Cytochrome c oxidase (CcO) is crucial for cellular respiration.
• Understanding CcO structural dynamics is key to elucidating its function.

Purpose of the Study:

• To apply Attenuated Total Reflection (ATR) spectroscopy for studying CcO structural changes.
• To record reaction-induced infrared difference spectra of CcO under controlled conditions.

Main Methods:

• Utilized ATR spectroscopy for vibrational difference spectra of CcO.
• Induced reduction via flow-exchange of aqueous buffer.
• Prepared CcO films from Rhodobacter sphaeroides and beef heart mitochondria reconstituted with lipid.

Main Results:

• ATR method successfully recorded vibrational difference spectra of CcO.
• CcO films retained full functionality, confirmed by visible and time-resolved vibrational spectroscopy.
• Demonstrated sufficient signal/noise to probe vibrational changes in individual residues.

Conclusions:

• Perfusion-induced Fourier-transform infrared difference spectroscopy is effective for complex enzymes like CcO.
• ATR spectroscopy provides a powerful tool for investigating enzyme mechanisms under diverse conditions.