Question

Two biochemistry students are about to use mitochondria isolated from rat liver for an experiment on oxidative phosphorylation. The directions for the experiment specify addition of purified cytochrome \(c\) from any source to the reaction mixture. Why is the added cytochrome \(c\) needed? Why does the source not have to be the same as that of the mitochondria?

Short answer

Added cytochrome c is required for electron transfer in oxidative phosphorylation. Its source can be any organism, as cytochrome c is highly conserved.

Step-by-step solution

- Understand cytochrome c's role

Cytochrome c is a vital component of the electron transport chain (ETC) in mitochondria. It acts as an electron carrier, transferring electrons between Complex III (cytochrome bc1 complex) and Complex IV (cytochrome c oxidase). This transfer is crucial for the process of oxidative phosphorylation, where the energy from electrons is used to produce ATP.

- Importance of adding cytochrome c

In isolated mitochondrial experiments, the endogenous cytochrome c might be lost or become inactive. Adding purified cytochrome c ensures that the electron transport chain can function properly, facilitating the transfer of electrons and subsequent production of ATP.

- Source of cytochrome c

Cytochrome c is highly conserved across different species, meaning its structure and function are very similar regardless of the source. Thus, cytochrome c from different organisms can effectively substitute the rat liver cytochrome c in oxidative phosphorylation experiments, maintaining the electron transfer function.

Key concepts

cytochrome c function

Cytochrome c is crucial in the process of oxidative phosphorylation. It is part of the electron transport chain (ETC), a series of protein complexes located in the inner mitochondrial membrane. Cytochrome c acts as an electron carrier. It transfers electrons from Complex III (cytochrome bc1 complex) to Complex IV (cytochrome c oxidase). This transfer is vital for the continuation of the ETC.
Without cytochrome c, the electrons would not efficiently move from one complex to the next, disrupting the chain and halting ATP production. In experiments involving isolated mitochondria, cytochrome c might be lost or denatured. Adding purified cytochrome c ensures that the ETC functions correctly. This process ensures the efficient production of ATP, which cells use as a primary energy source.
The reason the source of cytochrome c doesn't matter much is that this protein is highly conserved. Whether you get cytochrome c from a plant or an animal, its structure and function are very similar. This universality allows scientists to use cytochrome c from various sources in their experiments, as it will work effectively within the rat liver mitochondria.

mitochondrial electron transport chain

The mitochondrial electron transport chain (ETC) is a series of protein complexes and molecules that transfer electrons through a membrane within mitochondria. The ETC is essential for oxidative phosphorylation, the process by which cells produce ATP.
Located in the inner mitochondrial membrane, the ETC comprises four main protein complexes (I-IV). Electrons are transferred between these complexes through carriers such as coenzyme Q and cytochrome c. Here is a quick guide to the electron transfer process:

• Complex I transfers electrons from NADH to coenzyme Q.
• Complex II transfers electrons from succinate to coenzyme Q.
• Coenzyme Q passes electrons to Complex III.
• Complex III transfers electrons to cytochrome c.
• Cytochrome c then delivers electrons to Complex IV.
• Complex IV transfers electrons to oxygen, forming water.

As electrons move through these complexes, protons are pumped from the mitochondrial matrix to the intermembrane space. This creates a proton gradient across the inner mitochondrial membrane, which is crucial for ATP synthesis.
Without a functional ETC, the cell cannot produce enough ATP to meet its energy needs. Understanding how the ETC works is key to understanding cellular respiration and energy production.

ATP production

ATP, or adenosine triphosphate, is the energy currency of the cell. Cells produce ATP through a process called oxidative phosphorylation, which occurs in the mitochondria. The electron transport chain (ETC) plays a central role in this process.
As electrons move through the ETC, protons are pumped across the inner mitochondrial membrane, creating a proton gradient. This gradient stores potential energy. ATP production occurs in the final stage called chemiosmosis, facilitated by an enzyme called ATP synthase.
Here’s how ATP synthesis works:

• Protons flow back into the mitochondrial matrix through ATP synthase, driven by the proton gradient.
• ATP synthase uses the energy from this proton flow to convert ADP (adenosine diphosphate) and inorganic phosphate (Pi) into ATP.
• This process of converting ADP to ATP is known as phosphorylation.

Without sufficient electron flow through the ETC or if the proton gradient is disrupted, ATP synthesis would be inefficient or cease entirely. Thus, each component of the ETC and the proton gradient is crucial for the cell’s energy production.
Understanding ATP production helps in grasping how cells generate the energy needed for various biological processes, from muscle contraction to active transport of molecules across membranes.