EPR signals from cytochrome c oxidase

Area of Science:

• Biochemistry
• Biophysics
• Enzyme kinetics

Background:

• Cytochrome c oxidase (EC 1.9.3.1) is a crucial enzyme in cellular respiration.
• Electron paramagnetic resonance (EPR) spectroscopy is vital for studying paramagnetic centers in enzymes.
• Previous EPR studies of cytochrome c oxidase may have contained integration and simulation errors.

Purpose of the Study:

• To characterize the major EPR signals of native and reduced beef heart cytochrome c oxidase.
• To identify and quantify different copper (Cu2+) and heme species within the enzyme.
• To correct and refine EPR signal integration and simulation methodologies.

Main Methods:

• Electron paramagnetic resonance (EPR) spectroscopy at various frequencies (e.g., 35 GHz).
• Analysis of resonance parameters, signal components, and integrated intensities.
• Enzyme titration experiments using ascorbate and cytochrome c.

Main Results:

• The Cu2+ EPR signal comprises at least three components: one inactive and two active forms.
• A novel, cytochrome c-unreducible Cu2+ species (approx. 20% of detectable Cu) was observed.
• Low-spin heme signals showed no interaction with other paramagnetic centers.
• High-spin heme signals consisted of at least three species (axial and rhombic types).
• A new integration method for high-spin heme EPR signals was developed.

Conclusions:

• The study refines the understanding of EPR signal contributions from different copper and heme centers in cytochrome c oxidase.
• Identified inhomogeneity in enzyme preparations and previously unobserved copper species.
• The developed integration method improves the accuracy of EPR signal quantification.