How many NADH are produced in glycolysis?
Where glycolysis makes NADH, and what happens to it next
Matic Broz, PhD7 min
Glycolysis produces 2 NADH per glucose molecule. Both form at step 6, where the enzyme glyceraldehyde 3-phosphate dehydrogenase oxidizes glyceraldehyde 3-phosphate and passes two electrons to NAD⁺. Glucose is split into two of these three-carbon sugars, so the step runs twice. Glycolysis consumes no NADH and makes no FADH₂, which makes 2 both the gross and the net count.
Those two NADH are a small share of what glucose eventually yields. Complete aerobic breakdown of one glucose produces 10 NADH: 2 in glycolysis, 2 in pyruvate oxidation and 6 in the citric acid cycle. Glycolytic NADH is still the one that varies most in value. With oxygen, its electrons are worth about 3 or 5 ATP for the pair, depending on how they reach the mitochondrion. Without oxygen, fermentation spends them to regenerate NAD⁺ and gains no ATP at all.
Which step of glycolysis produces NADH?
NADH is produced only at step 6 of glycolysis, the first reaction of the payoff phase. Glyceraldehyde 3-phosphate dehydrogenase oxidizes the aldehyde group of glyceraldehyde 3-phosphate, attaches an inorganic phosphate and transfers two electrons to NAD⁺, forming NADH and 1,3-bisphosphoglycerate.[1][2]
No oxygen takes part. The sugar is oxidized because NAD⁺ removes electrons from it, and the reaction releases enough energy to fill two stores at once. Some stays with the electrons in NADH, and some goes into the new high-energy phosphate bond of 1,3-bisphosphoglycerate, which makes the pathway's first ATP at step 7.[1]
The doubling happens two steps earlier. Aldolase splits six-carbon fructose 1,6-bisphosphate into dihydroxyacetone phosphate and glyceraldehyde 3-phosphate, and triose phosphate isomerase converts the dihydroxyacetone phosphate into a second glyceraldehyde 3-phosphate.[2] Each of the two molecules then passes through step 6, so one glucose gives 2 NADH.[3]
Summed over all ten steps, glycolysis is:
Glucose + 2 NAD⁺ + 2 ADP + 2 inorganic phosphate → 2 pyruvate + 2 NADH + 2 H⁺ + 2 ATP + 2 water
The ATP count in this equation is net: the pathway spends 2 ATP in the investment phase and makes 4 in the payoff phase.[2] The NADH count needs no such correction, since no step of glycolysis uses NADH.
How many NADH and FADH₂ does each stage of cellular respiration produce?
Aerobic respiration of one glucose produces 10 NADH and 2 FADH₂. Glycolysis makes 2 NADH in the cytosol. The pyruvate dehydrogenase complex in the mitochondrion then converts each pyruvate into acetyl-CoA and carbon dioxide, making one NADH per pyruvate, or 2 per glucose.[1]
The citric acid cycle makes the rest. Each turn oxidizes one acetyl group and produces 3 NADH, 1 FADH₂ and 1 GTP.[1] Isocitrate dehydrogenase, the α-ketoglutarate dehydrogenase complex and malate dehydrogenase each reduce one NAD⁺, while succinate dehydrogenase reduces FAD to FADH₂.[4] One glucose supplies two acetyl groups, so the cycle turns twice and produces 6 NADH and 2 FADH₂.
| Stage | Location | Enzymes that form the carriers | NADH | FADH₂ |
|---|---|---|---|---|
| Glycolysis | Cytosol | Glyceraldehyde 3-phosphate dehydrogenase | 2 | 0 |
| Pyruvate oxidation | Mitochondrial matrix | Pyruvate dehydrogenase complex | 2 | 0 |
| Citric acid cycle | Mitochondrial matrix | Isocitrate, α-ketoglutarate and malate dehydrogenases (NADH); succinate dehydrogenase (FADH₂) | 6 | 2 |
| Total | 10 | 2 |
Glycolysis therefore contributes 2 of the 10 NADH, or 20%, and the Krebs cycle contributes 60%. Questions that ask for the cycle's NADH "per turn" expect 3; per pyruvate, counting the pyruvate dehydrogenase step, the answer is 4. Of the 12 electron carriers from one glucose, only the 2 glycolytic NADH are made outside the mitochondrion, and that location decides what they are worth.
What happens to the NADH from glycolysis?
Glycolytic NADH has two possible fates. With oxygen, its electrons are passed into the mitochondrion and on to oxidative phosphorylation. Without enough oxygen, the cell spends the NADH in the cytosol to regenerate NAD⁺. Either way the NAD⁺ must come back, because step 6 cannot run without it and the payoff phase of glycolysis would stall.[2]
Cells keep NAD⁺ well in excess of NADH outside the mitochondrion. Yang and Sauve report free NAD⁺/NADH ratios of about 60 to 700 in eukaryotic cells, against about 7 to 10 estimated for mitochondria, and describe the two routes that hold the cytoplasmic ratio high: shuttles into the mitochondrion and lactate dehydrogenase.[3]
With oxygen: shuttles into the mitochondrion
The inner mitochondrial membrane is impermeable to NADH and NAD⁺, so glycolytic NADH never enters the mitochondrion itself. Two shuttles carry its reducing equivalents across instead: the malate-aspartate shuttle and the glycerol 3-phosphate shuttle.[5]
The malate-aspartate shuttle regenerates NADH inside the matrix, where its electrons enter the respiratory chain at complex I like any other mitochondrial NADH. The glycerol 3-phosphate shuttle hands the electrons to the chain without passing through complex I, at the same level as electrons from succinate, so they pump fewer protons across the membrane.[6] In Hinkle's review of measured P/O ratios, electrons from NADH-linked substrates support about 2.5 ATP per pair and electrons from succinate about 1.5.[7]
| Route | Electrons enter the respiratory chain at | ATP per NADH | ATP from 2 NADH | Older accounting |
|---|---|---|---|---|
| Malate-aspartate shuttle | Complex I (as matrix NADH) | About 2.5 | About 5 | 6 |
| Glycerol 3-phosphate shuttle | Ubiquinone, bypassing complex I | About 1.5 | About 3 | 4 |
| Fermentation (no oxygen) | None | 0 | 0 | 0 |
Which shuttle a cell uses depends on the tissue. In the brain, the malate-aspartate shuttle is considered the main route, particularly in neurons.[5] The choice is why textbooks give a complete ATP yield of cellular respiration of either 30 or 32 per glucose, and why older sources that credit each NADH with 3 ATP give 36 or 38. Counting glycolysis's own 2 ATP, the pathway is worth about 5 to 7 ATP per glucose once its NADH is oxidized aerobically.
Without oxygen: fermentation regenerates NAD⁺
When oxygen is short, as in vigorously contracting skeletal muscle, the pyruvate and the NADH stay in the cytosol. Lactate dehydrogenase uses the NADH to reduce pyruvate to lactate,[3] while yeast convert pyruvate into ethanol and carbon dioxide. In both cases the NADH gives up its electrons and becomes NAD⁺ again, which keeps glycolysis running.[1]
The net NADH from glycolysis plus fermentation is therefore zero, and the cell keeps only the 2 ATP made directly by glycolysis. The electrons that could have produced another 3 to 5 ATP leave the cell in lactate or ethanol instead.
Why glycolytic NADH matters more than its share
Glycolysis makes a fifth of the NADH from glucose, but it is the only NADH whose value depends on where the cell sends it. The 8 NADH made inside the mitochondria always enter at complex I and are worth about 20 ATP between them. The 2 from glycolysis are where cells differ: a neuron relying on the malate-aspartate shuttle draws about 5 ATP from them, a cell using the glycerol 3-phosphate shuttle about 3, and a muscle fibre short of oxygen none.
