Ligand binding to ferrocytochrome c at high pH.

Area of Science:

• Biochemistry
• Protein Chemistry

Background:

• Ferrocytochrome c is a key protein in electron transport.
• Cytochrome c's interaction with ligands like carbon monoxide (CO) is crucial for understanding its function.
• Previous studies indicated CO binding to ferrocytochrome c, but the conditions and nature of the second binding were unclear.

Purpose of the Study:

• To investigate the binding of two carbon monoxide (CO) molecules to ferrocytochrome c at high pH.
• To elucidate the conditions under which the second CO molecule binds and its spectral properties.
• To understand the role of protein structure (denaturation and insolubilization) in CO binding.

Main Methods:

• Spectroscopic analysis (static titrations, flash-photolysis) to monitor CO binding.
• Utilizing modified cytochrome c forms: carboxymethyl cytochrome c and insoluble cytochrome c.
• Comparative ligand studies using cyanide (CN-) instead of CO.

Main Results:

• Ferrocytochrome c binds two CO molecules at pH 14.
• The second CO molecule binds only to denatured cytochrome c.
• This second CO binding event is spectrally silent and inhibited by protein insolubilization.

Conclusions:

• A model is proposed explaining the two-step CO binding mechanism.
• Protein denaturation is essential for the second CO molecule's binding.
• Protein structure, specifically insolubilization, prevents the second CO binding, suggesting conformational changes are involved.