Oxidative phosphorylation

Oxygen is a critical molecule for a process called oxidative phosphorylation. This two-step process uses electron transport chains and chemiosmosis to generate energy in the form of adenosine triphosphate (ATP). ATP is a major energy currency for active cells. Its synthesis is critical for the normal functioning of processes such as muscle contraction and active transport, to name a few. Oxidative phosphorylation takes place in the mitochondria, specifically in the inner membrane. The abundance of these organelles in particular cells is a good indication of how metabolically active they are!

Fig. 1 - The structure of ATP

Oxidative phosphorylation definition

Oxidative phosphorylation occurs only in the presence of oxygen and is therefore involved in aerobic respiration. Oxidative phosphorylation produces the most ATP molecules compared to other glucose metabolic pathways involved in cellular respiration, namely glycolysis and the Krebs cycle.

The two most essential elements of oxidative phosphorylation include the electron transport chain and chemiosmosis. The electron transport chain comprises membrane-embedded proteins, and organic molecules that are divided into four main complexes labelled I to IV. Many of these molecules are located in the inner membrane of the mitochondria of eukaryotic cells. This is different for prokaryotic cells, such as bacteria, whereby the electron transport chain components are instead located in the plasma membrane. As its name suggests, this system transports electrons in a series of chemical reactions called redox reactions.

Structure of mitochondria

This organelle has an average size of 0.75-3μm² and is composed of a double membrane, the outer mitochondrial membrane and the inner mitochondrial membrane, with an intermembrane space between them. Tissues such as the heart muscle have mitochondria with particularly large numbers of cristal because they must produce a lot of ATP for muscle contraction. There are around 2000 mitochondria per cell, which makes up approximately 25% of the cell volume. Located in the inner membrane are the electron transport chain and ATP synthase. Thus, they are referred to as the 'powerhouse' of the cell.

Mitochondria contain cristae, which are highly folded structures. Cristae increase the surface to volume ratio available for oxidative phosphorylation, meaning the membrane can hold a greater amount of electron transport protein complexes and ATP synthase than if the membrane was not highly convoluted. In addition to oxidative phosphorylation, the Krebs cycle also occurs in the mitochondria, specifically in the inner membrane known as the matrix. The matrix contains the Krebs cycle's enzymes, DNA, RNA, ribosomes, and calcium granules.

Oxidative phosphorylation diagram

Visualising oxidative phosphorylation can be really helpful in remembering the process and steps involved. Below is a diagram depicting oxidative phosphorylation.

Fig. 2 - Oxidative phosphorylation diagram

Oxidative phosphorylation process and steps

The synthesis of ATP via oxidative phosphorylation follows four main steps:

• Transport of electrons by NADH and FADH₂
• Proton pumping and electron transfer
• Formation of water
• ATP synthesis

Transport of electrons by NADH and FADH₂

NADH and FADH₂ (also referred to as reduced NAD and reduced FAD) are made during the earlier stages of cellular respiration in glycolysis, pyruvate oxidation and the Krebs cycle. NADH and FADH₂ carry hydrogen atoms and donate the electrons to molecules near the start of the electron transport chain. They subsequently revert to the coenzymes NAD⁺ and FAD in the process, which are then reused in early glucose metabolic pathways.

NADH carries electrons at a high energy level. It transfers these electrons to Complex I, which harnesses the energy released by the electrons moving through it in a series of redox reactions to pump protons (H⁺) from the matrix to the intermembrane space.

Meanwhile, FADH₂ carries electrons at a lower energy level and therefore does not transport its electrons to Complex I but to Complex II, which does not pump H⁺ across its membrane.

Proton pumping and electron transfer

Electrons go from a higher to a lower energy level as they move down the electron transport chain, releasing energy. This energy is used to actively transport H⁺ out of the matrix and into the intermembrane space. As a result, an electrochemical gradient is created, and H⁺ accumulate within the intermembrane space. This accumulation of H⁺ makes the intermembrane space more positive while the matrix is negative.

Formation of water

When the electrons reach Complex IV, an oxygen molecule will accept H⁺ to form water in the equation:

2H⁺ + $\frac{1}{2}$O₂ $\underset{}{\to }$ H₂O

ATP synthesis

H⁺ ions that have accumulated in the intermembrane space of the mitochondria flow down their electrochemical gradient and back into the matrix, passing through a channel protein called ATP synthase. ATP synthase is also an enzyme that uses the diffusion of H+ down its channel to facilitate the binding of ADP to Pi to generate ATP. This process is commonly known as chemiosmosis, and it produces over 80% of ATP made during cellular respiration.

In total, cellular respiration produces between 30 and 32 molecules of ATP for each glucose molecule. This produces a net of two ATP in glycolysis and two in the Krebs cycle. Two net ATP (or GTP) is produced during glycolysis and two during the citric acid cycle.

To produce one molecule of ATP, 4 H⁺ must diffuse through ATP synthase back into the mitochondrial matrix. NADH pumps 10 H⁺ into the intermembrane space; therefore, this equates to 2.5 molecules of ATP. FADH₂, on the other hand, only pumps out 6 H⁺, meaning only 1.5 molecules of ATP are produced. For every glucose molecule, 10 NADH and 2 FADH₂ are produced in previous processes (glycolysis, pyruvate oxidation and the Krebs cycle), meaning oxidative phosphorylation produces 28 molecules of ATP.

Oxidative phosphorylation products

Oxidative phosphorylation generates three main products:

• ATP
• Water
• NAD⁺ and FAD

ATP is produced due to the flow of H⁺ through ATP synthase. This is primarily driven by chemiosmosis which uses the electrochemical gradient between the intermembrane space and mitochondrial matrix. Water is produced at Complex IV, where atmospheric oxygen accepts electrons and H⁺ to form water molecules.

In the beginning, we read that NADH and FADH₂ deliver electrons to the proteins in the electron transport chain, namely Complex I and Complex II. When they release their electrons, NAD⁺ and FAD are regenerated and can be recycled back into other processes such as glycolysis, where they act as coenzymes.