Ascorbate reduction of horse heart cytochrome c. A zero-energy reduction reaction.

This research investigates how ascorbate, a type of vitamin C, reduces horse heart ferricytochrome c, an important protein in energy creation, under different conditions such as varying temperatures and pH levels. The finding suggests at lower temperatures, the reduction process occurs with near-zero activation energy and by a process known as tunneling.

Experimental Design

• The ascorbate reduction of horse heart cytochrome c was observed in a 0.05 M phosphate + 0.25 M sodium sulfate solution with a pH of 7.3 at temperatures ranging from 12-36 degrees Celsius.
• A two-step mechanism—binding of ascorbate to cytochrome c followed by reduction—was used to analyze the data.
• A stopped-flow technique, a method used to monitor ultra-rapid reactions, was used to perform the experiment at an alkaline pH of 8.4.

Results

• At temperatures up to about 26 degrees C, the first-order reduction constant remained indifferent to temperature changes, implying a zero or near-zero activation energy for the process. The activation energy suggests the energy input required to start a chemical reaction.
• Above 26 degrees C, the reduction constant becomes temperature-dependent, increasing with temperature and displaying an activation energy of about 35 kcal/mol.
• The ascorbate-cytochrome c complex’s stability increases between the temperature window of 12-26 degrees C, featuring an enthalpy and entropy change of about 3 kcal/mol and 32 e.u., respectively. Above this range, the complex’s stability decreases.
• At pH 8.4, the reduction reaction shows a biphasic behavior, consistent with the proposed two-step mechanism. A pH increase of 1.1 units boosts the first-order reduction constant by a factor of six and decreases the stability constant to about a quarter of its initial value.
• At pH 8.4, the slow phase of reaction is independent of ascorbate, exhibiting a rate constant of 0.043 +/- 0.006 s-1.

Final Conclusions

• The study concludes that the ascorbate reduction process of cytochrome c under temperatures up to 26 degrees C happens via a quantum mechanical process known as tunneling and shows zero or near-zero activation energy.
• The observed reduction constant is in line with this tunneling conclusion, suggesting a tunneling distance of about 16 A, and an energy barrier to overcome for the reaction to occur is about 35 kcal/mol.