Aerobic Cellular Respiration

Stage 1: Glycolysis. Glycolysis ("sugar splitting") is a sequence of ten enzyme-catalyzed reactions in the cytosol that splits one 6-carbon glucose into two 3-carbon pyruvate molecules. It needs no oxygen and no organelles, which is why nearly every living cell can do it and why it is thought to be one of the oldest metabolic pathways. It has two phases. In the energy-investment phase, 2 ATP are spent to phosphorylate glucose, first to glucose-6-phosphate and then to fructose-1,6-bisphosphate (the second phosphorylation is catalyzed by phosphofructokinase). The phosphates make the sugar unstable and trap it inside the cell, and the 6-carbon sugar is split into two 3-carbon molecules of G3P (glyceraldehyde-3-phosphate). In the energy-payoff phase, each G3P is oxidized by ${\text{NAD}}^{+}$ (making NADH), and its phosphate groups are passed to ADP by substrate-level phosphorylation, giving 2 ATP per G3P. Totals per glucose: 4 ATP made − 2 ATP invested = net 2 ATP, plus 2 NADH and 2 pyruvate.

Stages 2 and 3: Pyruvate oxidation and the Krebs cycle. If oxygen is available, pyruvate is actively transported into the mitochondrial matrix. In pyruvate oxidation, a large enzyme complex removes one carbon as ${\text{CO}}_2$, oxidizes the remaining 2-carbon piece (reducing ${\text{NAD}}^{+}$ to NADH) and attaches it to coenzyme A, making acetyl-CoA. This happens twice per glucose. The Krebs cycle (also called the citric acid cycle) then accepts acetyl-CoA. The 2-carbon acetyl group joins a 4-carbon molecule, oxaloacetate, to form 6-carbon citrate. Over the next several steps, citrate is oxidized and loses two carbons as ${\text{CO}}_2$, and the 4-carbon oxaloacetate is regenerated, ready for the next acetyl group; that is why it is a cycle. Each turn yields 3 NADH, 1 FADH₂, 1 ATP (by substrate-level phosphorylation) and 2 CO₂. Since each glucose produces two acetyl-CoA, the cycle turns twice per glucose. By the end of the Krebs cycle all six carbons of glucose have left as ${\text{CO}}_2$, yet only 4 ATP have been made. The energy is now sitting in 10 NADH and 2 ${\text{FADH}}_2$.

Stage 4: Electron transport and chemiosmosis. The electron transport chain (ETC) is a series of protein complexes and mobile carriers embedded in the inner mitochondrial membrane, which is folded into cristae to fit more of them. NADH donates its two electrons to the first complex (NADH dehydrogenase); ${\text{FADH}}_2$ donates its electrons to a later complex, skipping the first. The electrons then pass from carrier to carrier (through ubiquinone, cytochrome complexes and cytochrome c), each carrier more electronegative than the last, so the electrons lose free energy at each step. Three of the complexes use that released energy as proton pumps, actively moving ${\text{H}}^{+}$ from the matrix into the intermembrane space. At the end of the chain, oxygen is the final electron acceptor: $\frac{1}{2}{\text{O}}_2+2e^{-}+2{\text{H}}^{+}\to {\text{H}}_2\text{O}$. Without oxygen to remove electrons at the end, the whole chain backs up and stops.

Pumping creates an electrochemical gradient: the intermembrane space has a higher concentration of ${\text{H}}^{+}$ (lower pH) and more positive charge than the matrix. This gradient is stored potential energy, sometimes called the proton-motive force. The inner membrane is impermeable to ${\text{H}}^{+}$, so the only way back is through ATP synthase, a protein channel that works like a turbine: protons flowing down their gradient spin part of the enzyme, and that rotation drives the joining of ADP and ${\text{P}}_{\text{i}}$ into ATP. Using a proton gradient across a membrane to make ATP is called chemiosmosis (proposed by Peter Mitchell, who won a Nobel Prize for it in 1978). The whole process, electron transport plus chemiosmosis, is oxidative phosphorylation, because the ATP is made using energy from the oxidation of NADH and ${\text{FADH}}_2$.

How many ATP per glucose? Because oxidative phosphorylation is coupled through a gradient rather than a fixed recipe, the ATP count per NADH is not a whole number. Current measurements give about 2.5 ATP per NADH (roughly 10 ${\text{H}}^{+}$ pumped, and about 4 ${\text{H}}^{+}$ needed per ATP made and exported) and about 1.5 ATP per FADH₂ (it skips the first pump, so only about 6 ${\text{H}}^{+}$ are pumped). There is one more cost: the 2 NADH from glycolysis are made in the cytosol and cannot cross the inner membrane. In muscle and brain cells a shuttle passes their electrons to FAD inside the mitochondrion, so each is worth only 1.5 ATP; in liver and heart cells a different shuttle keeps them at 2.5. Some of the gradient is also spent importing pyruvate and phosphate, and some protons leak back through the membrane. Older sources used 3 ATP per NADH and 2 per ${\text{FADH}}_2$, giving 36–38 ATP; the modern estimate is about 30–32 ATP per glucose.

• Carbon dioxide — bubble the air from a flask of germinating seeds (or yeast in glucose solution) through limewater (calcium hydroxide solution). ${\text{CO}}_2$ forms insoluble calcium carbonate, turning the limewater milky. Alternatively use bromothymol blue: ${\text{CO}}_2$ forms carbonic acid, so the indicator changes from blue to green to yellow as the solution becomes more acidic.
• Heat — put germinating seeds in an insulated flask (a thermos) with a thermometer, and set up a second flask of seeds that have been boiled (killed) and disinfected so microbes cannot respire on them. Only the living seeds' flask warms up, showing that respiration releases energy as heat.
• Oxygen consumption — in a respirometer, potassium hydroxide absorbs the ${\text{CO}}_2$ produced, so any drop in gas volume (seen as a moving drop of coloured liquid in a capillary tube) is due to ${\text{O}}_2$ being used.
• Controls and variables — the dead seeds (or glass beads of equal volume) are the negative control; keep temperature, seed number and time the same. Record qualitative observations (colour change, cloudiness) as well as measurements (temperature, volume), and repeat trials for reliability.