Question

Transmembrane Movement of Reducing Equivalents Under aerobic conditions, extramitochondrial NADH must undergo oxidation by the mitochondrial respiratory chain. Consider a preparation of rat hepatocytes containing mitochondria and all the cytosolic enzymes. After the introduction of \(\left[4-{ }^{3} \mathrm{H}\right] \mathrm{NADH}\), radioactivity soon appears in the mitochondrial matrix. Conversely, no radioactivity appears in the matrix after the introduction of \(\left[7^{-14} \mathrm{C}\right]\) NADH. What do these observations reveal about the oxidation of extramitochondrial NADH by the respiratory chain?

Short answer

Electrons from NADH enter mitochondria via shuttles, not NADH itself.

Step-by-step solution

Understanding NADH Labeling

In this experiment, two types of labeled NADH are used: \(\left[4-^3 \text{H}\right] \text{NADH}\) and \(\left[7^{-14} \text{C}\right] \text{NADH}\). Each label corresponds to different atoms in the NADH molecule. When radioactivity is detected, it indicates the movement of the labeled compound or fragments of it.

Observing Radioactivity in Mitochondria

The observation shows that after introducing \(\left[4-^3 \text{H}\right] \text{NADH}\), radioactivity appears in the mitochondrial matrix, implying that some component of this NADH is being transported or transferred into the mitochondria.

Absence of Radioactivity in the Second Experiment

In contrast, when \(\left[7^{-14} \text{C}\right] \text{NADH}\) is used, no radioactivity is detected in the mitochondrial matrix. This indicates that the carbon of the NADH does not move into the mitochondria, confirming that the full molecule of NADH is not carried into the mitochondria.

Concluding the Type of Electron Shuttle

These results suggest that NADH itself does not enter the mitochondria. Instead, electrons from NADH are transferred across the membrane by a shuttle mechanism, such as the malate-aspartate or glycerol phosphate shuttles. These shuttles transfer reducing equivalents, changing the labeling pattern observed.

Key concepts

Mitochondrial Respiratory Chain

The mitochondrial respiratory chain, often called the electron transport chain, is pivotal for cellular respiration. It is located in the inner mitochondrial membrane and is responsible for the generation of ATP through oxidative phosphorylation.
The process involves a series of protein complexes and other molecules that transfer electrons from donors to acceptors through redox reactions.

• Complex I, known as NADH:ubiquinone oxidoreductase, begins the process by oxidizing NADH and transferring the electrons to ubiquinone, a fat-soluble carrier.
• This step is crucial for the later stages of electron transfer through Complexes II, III, and IV, ultimately reaching oxygen, which gets reduced to water.

Each step in the chain facilitates proton pumping into the intermembrane space, creating an electrochemical gradient. This gradient drives the synthesis of ATP, which is crucial for cellular energy requirements. Understanding this chain is essential for grasping how aerobic cells efficiently produce energy.

NADH Oxidation

NADH oxidation is a critical step in cellular respiration, providing the necessary reducing equivalents for the electron transport chain. NADH, produced in the cytosol during glycolysis and in the mitochondria during the citric acid cycle, must undergo oxidation to facilitate ATP production.
Oxidation of NADH involves transferring its electrons to the mitochondrial respiratory chain.

• First, NADH transfers electrons to Complex I in the mitochondrial membrane, where it's oxidized to NAD⁺.
• The electrons are then passed through the sequential series of electron carriers within the electron transport chain.

This process is not only essential for maintaining the cellular redox state but also for generating ATP efficiently. Moreover, the resistance to direct translocation of NADH into mitochondria indicates the necessity of shuttle systems to transport electrons from cytosolic NADH.

Electron Shuttle Mechanisms

Within cells, not all components can freely cross the mitochondrial membranes, leading to the innovation of electron shuttle mechanisms. These systems allow the transfer of reducing equivalents—primarily electrons—into the mitochondria without the need for the physical passage of NADH.
Two prominent shuttles are frequently discussed:

• Malate-Aspartate Shuttle: This shuttle facilitates the transfer of electrons from NADH by converting oxaloacetate to malate in the cytoplasm. Malate can be transported into the mitochondria, where it is reoxidized to oxaloacetate, releasing the electrons to the mitochondrial NAD⁺.
• Glycerol Phosphate Shuttle: This mechanism is more prevalent in muscle tissue. It transfers electrons from NADH to dihydroxyacetone phosphate, forming glycerol 3-phosphate, which can easily enter mitochondria. There, it donates the electrons to FAD, forming FADH₂ that enters the electron transport chain.

Understanding these mechanisms illuminates how cells efficiently manage and transfer energy even across impermeable barriers, maintaining the vital supply of ATP.