Where does the oxygen released in photosynthesis come from?

The click

From water, not from carbon dioxide. In the light reactions, water molecules are split using light energy, a step called photolysis that happens at photosystem II: 2H₂O → 4H⁺ + 4e⁻ + O₂. The electrons replace those lost by chlorophyll when it absorbs light, the H⁺ ions help build the proton gradient that drives ATP synthesis, and the O₂ is released as a by-product.

The CO₂ is used in a different stage, the Calvin cycle in the stroma, where its carbon and oxygen atoms are built into sugar, with some of the oxygen ending up in water.

The idea was first proposed by Cornelis van Niel, from his work on purple and green sulphur bacteria. These bacteria use H₂S instead of water as their hydrogen source and release sulphur, not oxygen, suggesting that in green plants the oxygen comes from the water. The proof came in 1941 from experiments by Ruben, Kamen and colleagues using the heavy isotope of oxygen, ¹⁸O: when the water was labelled, the oxygen released was labelled; when the CO₂ was labelled instead, it was not.

This is why the full equation is often written 6CO₂ + 12H₂O → C₆H₁₂O₆ + 6H₂O + 6O₂: twelve water molecules supply all twelve oxygen atoms in the six O₂ molecules. The shorter 6CO₂ + 6H₂O → C₆H₁₂O₆ + 6O₂ is balanced, but it hides where the oxygen comes from.