Oxidized (left) and reduced (right) Cyto c
In the body, cytochrome c is an important heme protein involved in the electron transport chain (ETC) and thus the body’s energy production, and is found in the inner membrane of mitochondria. It exists both in the oxidized and reduced state, which makes sense since its role in the ETC is to accept an electron from Complex III and transfer it to Complex IV via coupled oxidation/reduction (redox) reactions.
When a chemical is oxidized or reduced, that reaction forms half of a complete redox reaction. Each half of a redox reaction is called a half reaction, where an electron is either gained or lost. The oxidation of iron to rust is a half reaction, with the reduction of oxygen and water forming the other half of the complete redox interaction. One half reaction cannot proceed without its other half, so a molecule cannot be oxidized without another molecule being reduced (and vice versa).
The oxidation and reduction of cytochrome c can be quantified using UV-Vis spectroscopy (ultraviolet-visible spectroscopy), a technique that measure the absorbancy of samples in the visible, and near-visible, regions of light. The electrons in molecules will, when exposed to light, become excited to higher molecular orbitals. More easily excitable electrons absorb higher wavelengths of light due to the decreased energy difference in the ground state and excited state.
With cytochrome c, the reduced form has an absorbancy at 550nm, a characteristic not shared by the oxidized form. Light of wavelength 550nm falls in the green portion of the visible spectrum. The sample to the right, the ferrocytochrome c (reduced cytochrome c), thus has a different color than the sample to the left, the ferricytochrome c (oxidized cytochrome c). Both have similar absorbancies from 300 to 800 nm, with the exception of this peak at 550 nm, which explains the similar underlying orange hue. Using a number of calculations, namely the Nerst equation, we were able to find the midpoint reduction potential of cytochrome c (a measurement quantifying the ease of reducing cyto c in a redox reaction).
A version of this lab can be found in the publication by Craig and Nichols (2006) in the Journal of Chemical Education.