Into the Mitochondrion: Making ATP with Oxygen

Enticing clues - volcanic gases, vast iron ore sediments, and bubbles of ancient air trapped in amber – suggest dramatic changes during the history of earth’s atmosphere. Correlating these clues with the fossil record leads to two major conclusions: that early life evolved in the absence of oxygen, and that oxygen first appeared between 2 and 3 billion years ago (Figure 1) because of photosynthesis by bluegreen bacteria (Figure 2). The chemistry of cellular respiration reflects this history. Its first stage, glycolysis, is universal and does not use oxygen.

Absolutely dependent on oxygen gas, we find it difficult to imagine that its appearance must have been disastrous for the anaerobic organisms that evolved in its absence. But oxygen is highly reactive, and at first, its effect on evolution was so negative that some have named this period the “oxygen catastrophe.” However, as oxygen gradually formed a protective ozone layer, life rebounded. After the first organisms “discovered” how to use oxygen to their advantage – the diversity of aerobic organisms exploded. According to the endosymbiotic theory, engulfing of some of these aerobic bacteria led to eukaryotic cells with mitochondria, and multicellularity followed. Today, we live in an atmosphere which is 21% oxygen, and most of life follows glycolysis with the last two, aerobic stages of cellular respiration.

Figure 1: Oxygen has increased in the atmosphere throughout the history of the earth. Note the logarithmic scale, which indicates great increases after first photosynthesis and then land plants evolved. Related geological events: A = no oxidized iron; B = oxidized iron bands in seabed rock - evidence for \(O_2\) in the oceans; C= oxidized iron bands on land and ozone layer formation- evidence for \(O_2\) in the atmosphere.

Figure 2: Bubbles of oxygen appear at the surface above a mat of bluegreen bacteria in a freshwater pond. Studies of the fossil record and earth’s atmosphere suggest that life evolved before bacteria similar to these first added oxygen.

Recall the purpose of cellular respiration: to release energy from glucose to make ATP - the universal “currency” for cellular work. The following equation describes the overall process, although it summarizes many individual chemical reactions.

$$6O_2 + \stackrel{\hbox{C}_6 \hbox{H}_12 \hbox{O}_6}{{\scriptsize \text{Deliverable stored chemical energy}}} + 38\text{ADP} + 38\text{P}_i \xrightarrow{\stackrel{\text{enzymes}}{\text{mitochondria}}} \stackrel{\hbox{38ATP}}{{\scriptsize \text{Usable stored energy}}} + 6CO_2 + 6H_2 O$$

Once again, the first stage of this process, glycolysis, is ancient, universal, and anaerobic. In the cytoplasm of most cells, glycolysis breaks each 6-carbon molecule of glucose into two 3-carbon molecules of pyruvate. Chemical energy, which had been stored in the now broken bonds, is transferred to 2 ATP and 2 “hot hydrogens,” NADH.

The fate of pyruvate depends on the species and the presence or absence of oxygen. If oxygen is present to drive subsequent reactions, pruvate enters the mitochondrion, where the Krebs Cycle (Stage 2) and electron transport chain (Stage 3) break it down and oxidize it completely to \(CO_2\) and \(H_2 O\). The energy thus released builds many more ATP molecules, though of course some is lost as heat. Let’s explore the details of how mitochondria use oxygen to make more ATP from glucose by aerobic respiration.

The Krebs Cycle: Capturing Energy from Pyruvate

Aerobic respiration begins with the entry of pyruvate (product of glycolysis) into the mitochondria. We will follow the six carbons of the original glucose molecule, so we will consider two 3-carbon pyruvates. The fate of pyruvate’s energy and carbon atoms can be followed in the examples below:

1. Within the mitochondria, each pyruvate is broken apart and combined with a coenzyme known as CoA to form a 2-carbon molecule, Acetyl CoA, which can enter the Krebs Cycle. A single atom of carbon (per pyruvate) is “lost” as carbon dioxide. The energy released in this breakdown is captured in two “hot hydrogen” – NADH. See Figure 3. Fatty acids can also break down into Acetyl CoA. By this means, lipids, like carbohydrates, can be “burned” to make ATP using the Krebs Cycle.

Figure 3: After glycolysis, two 3-carbon pyruvates enter the mitochondrion, where they are converted to two 2-carbon acetyl CoenzymeA (CoA) molecules. Acetyl CoA then enters the Krebs Cycle. Note that the carbons removed become carbon dioxide, accounting for two of the six such end products of glucose oxidation. The energy released by this breakdown is carried by “hot hydrogen.”

1. The Krebs Cycle (Figure 4) begins by combining each Acetyl CoA with a four-carbon carrier molecule to make a 6-carbon molecule of citric acid (or citrate, its ionized form). For this reason, the Krebs Cycle, named for a scientist who worked out its details, is also called the Citric Acid Cycle.

2. The cycle carries citric acid through a series of chemical reactions which gradually release energy and capture it in several carrier molecules. For each Acetyl CoA which enters the cycle, 3 NAD+ are reduced to NADH, one molecule of FAD (yet another temporary energy carrier we haven’t met before) is reduced to FADH2, and one molecule of ATP (actually a precursor, GTP) is made. Study Figure 4 to locate each of these energy-capturing events.

3. Note what happens to carbon atoms (black dots in Figure 4). For each 2-carbon Acetyl CoA which enters the cycle, two molecules of carbon dioxide are released - complete breakdown of the original 6-carbon glucose molecule. The final step regenerates the original 4-carbon molecule which began the cycle, so that another Acetyl CoA can enter.

Figure 4: The Krebs or Citric Acid Cycle completes the breakdown of glucose begun in glycolysis. If oxygen is present, pyruvate enters the mitochondria and is converted to Acetyl CoA. Acetyl CoA enters the cycle by combining with 4-carbon oxaloacetate. Study the diagram to confirm that each turn of the cycle (two for each glucose) stores energy in \(3 NADH+H^+\), one \(FADH_2\), and one ATP (from GTP), and releases \(2CO_2\).

In summary, the Krebs Cycle completes the breakdown of glucose which began with glycolysis. Its chemical reactions oxidize all six of the original carbon atoms to \(CO_2\), and capture the energy released in 2 ATP, 6 NADH, and \(2FADH_2\). These energy carriers join the 2 ATP and 2 NADH produced in glycolysis and the 2 NADH produced in the conversion of 2 pyruvates to 2 Acetyl CoA.

At the conclusion of the Krebs Cycle, glucose is completely broken down, yet only four ATP have been produced. Moreover, although oxygen is required to drive the Krebs Cycle, the cycle’s chemical reactions do not themselves consume \(O_2\). The conclusion of cellular respiration – its “grand finale!” – produces the majority of the ATP.

Structure of the Mitochondrion: Key to Aerobic Respiration

As noted earlier, the aerobic phases of cellular respiration in eukaryotes occur within organelles called mitochondria. A detailed look at the structure of the mitochondrion (Figure 5) helps to explain its role in the last stage of respiration, the electron transport chain.

Figure 5: Mitochondria, organelles specialized to carry out aerobic respiration, contain an inner membrane folded into cristae, which form two separate kinds of compartments: inner membrane space and matrix. The Krebs Cycle takes place in the matrix. The electron transport chain is embedded in the inner membrane and uses both compartments to make ATP by chemiosmosis.

Two separate membranes form the mitochondrion. The inner membrane folds into cristae which divide the organelle into three compartments – intermembrane space (between outer and inner membranes), cristae space (formed by infoldings of the inner membrane), and matrix (within the inner membrane). The Krebs Cycle takes place within the matrix. The compartments are critical for the electron transport chain. Glycolysis occurs in the cytoplasm of the cell, with the products of glycolysis entering the mitochondria to continue cellular respiration.

The Electron Transport Chain: ATP for Life in the Fast Lane

At the end of the Krebs Cycle, energy from the chemical bonds of glucose is stored in diverse energy carrier molecules: four ATP, but also two \(\boldsymbol{FADH_2}\) and ten NADH. The primary task of the last stage of cellular respiration, the electron transport chain (ETC), is to transfer energy from these carriers to ATP, the “batteries” which power work within the cell.

Pathways for making ATP in stage 3 of aerobic respiration closely resemble the electron transport chains used in photosynthesis. In both ETCs, energy carrier molecules are arranged in sequence within a membrane so that energy-carrying electrons cascade from one to another, losing a little energy in each step. In both photosynthesis and aerobic respiration, the energy lost is harnessed to pump hydrogen ions into a compartment, creating an electrochemical or chemiosmotic gradient across the enclosing membrane. And in both processes, the energy stored in the chemiosmotic gradient is used to build ATP.

Figure 6: The third stage of photosynthesis uses the energy stored earlier in NADH and \(FADH_2\) to make ATP. Electron transport chains embedded in the inner membrane capture high-energy electrons from the carrier molecules and use them to concentrate hydrogen ions in the intermembrane space. Hydrogen ions flow down their electrochemical gradient back into the matrix through channels which capture their energy to convert ADP to ATP.

For aerobic respiration, the electron transport chain or “respiratory chain” is embedded in the inner membrane of the mitochondria (Figure 6). \(FADH_2\) and NADH (produced in glycolysis and the Krebs Cycle) donate high-energy electrons to energy carrier molecules within the membrane. As they pass from one carrier to another, the energy they lose is used to pump hydrogen ions into the intermembrane space, creating an electrochemical gradient. Hydrogen ions flow “down” the gradient – from outer to inner compartment – through an ion channel/enzyme, ATP synthase, which transfer their energy to ATP. Note the paradox that it requires energy to create and maintain a concentration gradient of hydrogen ions that are then used by ATP synthase to create stored energy (ATP). In broad terms, it takes energy to make energy. Coupling the electron transport chain to ATP synthesis with a hydrogen ion gradient is chemiosmosis, first described by Nobel laureate Peter D. Mitchell.

After passing through the ETC, low-energy electrons and low-energy hydrogen ions combine with oxygen to form water. Thus, oxygen’s role is to drive the entire set of ATP-producing reactions within the mitochondrion by accepting “spent” hydrogens. Oxygen is the final electron acceptor; no part of the process - from the Krebs Cycle through electron transport chain – can happen without oxygen.

The electron transport chain can convert the energy from one glucose molecule’s worth of \(FADH_2\) and \(NADH+H^+\) into as many as 34 ATP. When the four ATP produced in glycolysis and the Krebs Cycle are added, the total fits the overall equation for aerobic cellular respiration:

$$6O_2 + \stackrel{\hbox{C}_6 \hbox{H}_12 \hbox{O}_6}{{\scriptsize \text{Deliverable stored chemical energy}}} + 38\text{ADP} + 38\text{P}_i \xrightarrow{\stackrel{\text{enzymes}}{\text{mitochondria}}} \stackrel{\hbox{38ATP}}{{\scriptsize \text{Usable stored energy}}} + 6CO_2 + 6H_2 O$$

Aerobic respiration is complete. If oxygen is available, cellular respiration transfers the energy from one molecule of glucose to 38 molecules of ATP, releasing carbon dioxide and water as waste. “Deliverable” food energy has become energy which can be used for work within the cell – transport within the cell, pumping ions and molecules across membranes, and building large organic molecules. Can you see how this could lead to “life in the fast lane” compared to anaerobic respiration (glycolysis alone)?