Ion binding to cytochrome c studied by nuclear magnetic quadrupole relaxation

Area of Science:

• Biochemistry
• Biophysical Chemistry
• Nuclear Magnetic Resonance Spectroscopy

Background:

• Cytochrome c is a crucial protein in cellular respiration.
• Understanding ion binding to cytochrome c is vital for elucidating its function and structure.
• Chloride ion interactions with proteins can significantly impact their properties.

Purpose of the Study:

• To investigate the binding of chloride ions (35Cl-) to both oxidized and reduced forms of cytochrome c.
• To characterize the binding sites and affinities of chloride ions.
• To explore the influence of pH, temperature, and other ions on chloride binding.

Main Methods:

• Utilized 35Cl- Nuclear Magnetic Resonance (NMR) spectroscopy to measure transverse relaxation rates.
• Performed competition experiments with phosphate, iron hexacyanide, and cyanide.
• Studied the effects of varying concentrations of NaCl, sodium phosphate, iron hexacyanide, and sodium cyanide.
• Analyzed pH dependence using literature pK values.
• Employed 23Na+ NMR to investigate potential sodium ion binding.

Main Results:

• Demonstrated strong chloride binding sites on both oxidized and reduced cytochrome c, with higher affinity for the reduced form (ferrocytochrome c).
• Identified competition for these sites by iron hexacyanide and phosphate.
• Observed that cyanide binding reduces chloride binding to ferricytochrome c at neutral and alkaline pH.
• Found no evidence of sodium ion binding to cytochrome c using 23Na+ NMR.
• Fitted pH-dependent relaxation rates using known pK values.

Conclusions:

• Chloride ions bind to cytochrome c near the exposed heme edge.
• The surface structure and/or dynamics near the heme edge differ between oxidized and reduced cytochrome c.
• These findings provide insights into the molecular interactions governing cytochrome c function.