A spin label study of conformational changes in cytochrome c

Area of Science:

• Biochemistry
• Structural Biology
• Electron Paramagnetic Resonance (EPR) Spectroscopy

Background:

• Cytochromes c are vital electron transport proteins.
• Understanding their conformational dynamics is key to elucidating their function.
• Spin labeling combined with EPR offers a sensitive method to probe protein structure and dynamics.

Purpose of the Study:

• To investigate conformational changes in pig heart cytochrome c.
• To analyze the effects of ligand binding, redox state, and pH on protein structure.
• To map structural alterations using site-specific spin labels.

Main Methods:

• Site-specific spin labeling of pig heart cytochrome c at Met-65, Tyr-74, Lys-72, and Lys-73.
• Electron Spin Resonance (ESR) spectroscopy to monitor label mobility.
• Varying ligand (cyanide) and redox states of the heme.
• pH titration to identify conformational transitions.

Main Results:

• Cyanide binding at Met-80 significantly increased label mobility, indicating structural changes.
• Reduction of ferricytochrome c showed minimal spectral changes.
• Two pH-dependent conformational transitions were observed before alkaline denaturation (pK 9.3 and pK 11.1).
• Met-65 label detected the first transition; Tyr-74 and Lys-72(73) labels detected the second.

Conclusions:

• Ligand exchange at the heme's sixth coordination site induces conformational changes.
• These changes affect different regions of the cytochrome c molecule.
• The study provides insights into the structural flexibility of cytochromes c in response to environmental factors.