Oxygenic Photoreactivity in Photosystem II Studied by Rotating Ring Disk Electrochemistry

[Image: see text] Protein film photoelectrochemistry has previously been used to monitor the activity of photosystem II, the water-plastoquinone photooxidoreductase, but the mechanistic information attainable from a three-electrode setup has remained limited. Here we introduce the four-electrode rotating ring disk electrode technique for quantifying light-driven reaction kinetics and mechanistic pathways in real time at the enzyme–electrode interface. This setup allows us to study photochemical H(2)O oxidation in photosystem II and to gain an in-depth understanding of pathways that generate reactive oxygen species. The results show that photosystem II reacts with O(2) through two main pathways that both involve a superoxide intermediate to produce H(2)O(2). The first pathway involves the established chlorophyll triplet-mediated formation of singlet oxygen, which is followed by its reduction to superoxide at the electrode surface. The second pathway is specific for the enzyme/electrode interface: an exposed antenna chlorophyll is sufficiently close to the electrode for rapid injection of an electron to form a highly reducing chlorophyll anion, which reacts with O(2) in solution to produce O(2)(•–). Incomplete H(2)O oxidation does not significantly contribute to reactive oxygen formation in our conditions. The rotating ring disk electrode technique allows the chemical reactivity of photosystem II to be studied electrochemically and opens several avenues for future investigation.