Pulsed high-frequency EPR study on the location of carotenoid and chlorophyll cation radicals in photosystem II

When the primary electron-donation pathway from the water-oxidation complex in photosystem II (PS II) is inhibited, chlorophyll (Chl_Z and Chl_D), β-carotene (Car) and cytochrome b₅₅₉ are alternate electron donors that are believed to function in a photoprotection mechanism. Previous studies have demonstrated that high-frequency EPR spectroscopy (at 130 GHz), together with deuteration of PS II, yields resolved Car⁺ and Chl⁺ EPR signals (Lakshmi et al. J. Phys. Chem. B 2000, 104, 10 445-10 448). The present study describes the use of pulsed high-frequency EPR spectroscopy to measure the location of the carotenoid and chlorophyll radicals relative to other paramagnetic cofactors in Synechococcus lividus PS II. The spin-lattice relaxation rates of the Car⁺ and Chl⁺ radicals are measured in manganese-depleted and manganese-depleted, cyanide-treated PS II; in these samples, the non-heme Fe(II) is high-spin (S = 2) and low-spin (S = 0), respectively. The Car⁺ and Chl⁺ radicals exhibit dipolar-enhanced relaxation rates in the presence of high-spin (S = 2) Fe(II) that are eliminated when the Fe(II) is low-spin (S = 0). The relaxation enhancements of the Car⁺ and Chl⁺ by the non-heme Fe(II) are smaller than the relaxation enhancement of Tyr_D· and P₈₆₅⁺ by the non-heme Fe(II) in PS II and in the reaction center from Rhodobacter sphaeroides, respectively, indicating that the Car⁺-Fe(II) and Chl⁺-Fe(II) distances are greater than the known Tyr_D·-Fe(II) and P₈₆₅⁺-Fe(II) distances. The Car⁺ radical exhibits a greater relaxation enhancement by Fe(II) than the Chl⁺ radical, consistent with Car being an earlier electron donor to P₆₈₀⁺ than Chl. On the basis of the distance estimates obtained in the present study and by analogy to carotenoid-binding sites in other pigment-protein complexes, possible binding sites are discussed for the Car cofactors in PS II. The relative location of Car⁺ and Chl⁺ radicals determined in this study provides valuable insight into the sequence of electron transfers in the alternate electron-donation pathways of PS II.