Hydrogen-isotope exchange of oxidized and reduced cytochrome c. A comparison of mass spectrometry and infrared methods

Area of Science:

• Biochemistry
• Protein Dynamics
• Spectroscopy

Background:

• Cytochrome c is a crucial protein in cellular respiration.
• Understanding its structural dynamics is key to elucidating its function.
• Hydrogen-deuterium exchange is a powerful technique for probing protein structure and dynamics.

Purpose of the Study:

• To investigate hydrogen-deuterium exchange in horse heart cytochrome c.
• To compare the exchange kinetics between the ferric and ferro redox states.
• To characterize the structural implications of redox state changes.

Main Methods:

• Mass spectrometry was employed to quantify hydrogen exchange.
• Infrared spectroscopy was used to analyze peptide hydrogen exchange and conformational changes.
• Experiments were conducted in 2H2O solutions at 20°C and pH 7.

Main Results:

• Significant differences in hydrogen exchange rates were observed between ferricytochrome c and ferrocytochrome c.
• Mass spectrometry revealed varying numbers of unexchanged, slowly exchanging, and rapidly exchanging hydrogens.
• Infrared spectroscopy indicated that 30% and 40% of peptide hydrogens are protected in ferric and ferrocytochrome c, respectively, suggesting increased rigidity in the reduced form.

Conclusions:

• The redox state of cytochrome c significantly impacts its hydrogen exchange dynamics and conformational flexibility.
• Ferrocytochrome c exhibits greater structural rigidity compared to ferricytochrome c.
• These findings provide insights into the structural basis of cytochrome c function and redox-dependent conformational changes.