Characterization of the secondary electron-transfer pathway intermediates of photosystem II containing low-potential cytochrome b₅₅₉

β-carotene (Car) and chlorophyll (Chl) function as secondary electron donors in photosystem II (PS II) under conditions, such as low temperature, when electron donation from the O₂-evolving complex is inhibited. In prior studies of the formation and decay of Car^•+ and Chl ^•+ species at low temperatures, cytochrome b₅₅₉ (Cyt b₅₅₉) was chemically oxidized prior to freezing the sample. In this study, the photochemical formation of Car^•+ and Chl ^•+ is characterized at low temperature in O₂-evolving Synechocystis PS II treated with ascorbate to reduce most of the Cyt b ₅₅₉. Not all of the Cyt b₅₅₉ is reduced by ascorbate; the remainder of the PS II reaction centers, containing oxidized low-potential Cyt b₅₅₉, give rise to Car^•+ and Chl^•+ species after illumination at low temperature that are characterized by near-IR spectroscopy. These data are compared to the measurements on ferricyanide-treated O₂-evolving Synechocystis PS II in which the Car^•+ and Chl^•+ species are generated in PS II centers containing mostly high- and intermediate-potential Cyt b₅₅₉. Spectral differences observed in the ascorbate-reduced PS II samples include decreased intensity of the Chl^•+ and Car^•+ absorbance peaks, shifts in the Car^•+ absorbance maxima, and lack of formation of a 750 nm species that is assigned to a Car neutral radical. These results suggest that different spectral forms of Car are oxidized in PS II samples containing different redox forms of Cyt b₅₅₉, which implies that different secondary electron donors are favored depending on the redox form of Cyt b₅₅₉ in PS II.