The effect of binding ions on the oxidation of horse heart ferrocytochrome c.

The research explores how different binding ions affect the oxidation speed of horse heart ferrocytochrome c, a protein, by potassium ferricyanide at a constant ionic strength.

Studying the Ion Effect on Oxidation Speed

• The study focused on determining the impact various binding ions have on the oxidation process of ferrocytochrome c, a horse heart protein when oxidized with potassium ferricyanide.
• The ionic strength – the concentration of ions in a solution – was kept constant for this study using a Tris-cacodylate buffer, with a pH level of 7.0.
• Within this buffer, either the Tris or cacodylate ion was replaced with the binding ion that was being studied, such as chloride, phosphate, potassium, or picrate.

Observations from the Ion Substitution

• It was observed that changing cacodylate to chloride at an ionic strength of 0.194 M at a temperature of 24°C led to a decline in the bimolecular oxidation rate constant – the speed at which the reaction occurs.
• Similar decreases were also noted when Tris was substituted for potassium or cacodylate was replaced by phosphate.
• The biggest decrease was seen when picrate was used instead of cacodylate at an ionic strength of 0.0485 M.

The Binding Model and Association Constants

• The collected data from studying varying ion concentrations aligned with a straightforward cooperative binding model, indicating that ions and the protein were working together during the oxidation process.
• The derived association constants for the chloride, potassium, and phosphate ions ranged from 2-20 M^(-1), while that of picrate was 500 M^(-1).
• The data suggest that each molecule of ferrocytochrome c bound to one ion, with the exception of picrate, which indicated two binding sites.

Interpretations and Hypotheses

• The researchers hypothesize that the picrate ion binds near the heme edge – the border of the iron-containing part of the protein – exposed to the solvent.
• They also propose that the electron transfer reaction of ferricyanide, which drives the oxidation process, occurs in this region too.
• In contrast to picrate, the other ions likely bind to sites located further away from the heme edge. The researchers believe these ions influence the oxidation process by changing the protein’s solvent shell – the layer of solvent particles that interacts directly with the protein.
• A notable observation was that replacing H2O with D2O (heavy water) led to approximately a 50% decrease in the oxidation rate constant, supporting the idea that the solvent shell plays a significant role in the oxidation process.