Cytochrome c/cytochrome c oxidase interaction. Direct structural evidence for conformational changes during enzyme...

Area of Science:

• Biochemistry
• Enzyme kinetics
• Protein-protein interactions

Background:

• Cytochrome c oxidase (CcO) is a key enzyme in cellular respiration.
• Understanding CcO substrate interactions is crucial for elucidating electron transport mechanisms.

Purpose of the Study:

• To investigate the interaction between CcO and its substrate, cytochrome c.
• To characterize covalent enzyme-substrate complexes and their electron transfer properties.

Main Methods:

• Formation of 1:1 covalent CcO-cytochrome c complexes under low ionic strength.
• Characterization using steady-state spectral analysis and stopped-flow kinetics.
• Electrophoresis and structural analysis to identify binding sites and involved residues.

Main Results:

• Covalent complexes formed with cytochrome c bound to subunit II (oxidized) or subunits II, VIb, and IV (steady-state reducing).
• Bound cytochrome c efficiently transferred electrons to the enzyme (t½ < 5 ms).
• Covalent complexes showed reduced catalytic activity with exogenous ferrocytochrome c compared to electrostatic complexes, indicating intramolecular electron transfer.

Conclusions:

• Electrons enter CcO via two independent pathways, involving different substrate binding sites.
• CcO likely cycles between two conformers during enzyme turnover.
• Specific residues in subunits IV and VIb may be involved in substrate binding and electron transfer.