Chlorophyll and carotenoid radicals in photosystem II studied by pulsed ENDOR

The stable carotenoid cation radical (Car(.+)) and chlorophyll cation radical (Chl(z)(.+)) in photosystem II (PS II) have been studied by pulsed electron nuclear double resonance (ENDOR) spectroscopy. The spectra were essentially the same for oxygen-evolving PS II and Mn-depleted PS II. The radicals were generated by illumination given at low temperatures, and the ENDOR spectra were attributed to Car(.+) and Chl(z)(.+) on the basis of their characteristic behavior with temperature as demonstrated earlier [Hanley et al. (1999) Biochemistry 38, 8189-8195]: i.e., (a) the Car(.+) alone was generated by illumination at less than or equal to 20 K, while Chl(z)(.+) alone was generated at 200 K, and (b) warming of the sample containing the Car(.+) to 200 K resulted in the loss of the signal attributable to Car(.+) and its replacement by a spectrum attributable to the Chl(z)(.+). A map of the hyperfine structure of Car(.+) in PS II and in organic solvent was obtained. The largest observed hyperfine splitting for Car(.+) in either environment was in the order of 8-9 MHz. Thus, the spin density on the cation is proposed to be delocalized over the carotenoid molecule. The pulsed ENDOR spectrum of Chl(z)(.+) was compared to that obtained from a Chl a cation in frozen organic solvent. The hyperfine coupling constants attributed to the beta -protons at position 17 and 18 are well resolved from Chl(z)(.+) in PS II (10.8 and 14.9 MHz) but not in Chl a(.+) in organic solvent (12.5 MHz). This suggests a more defined conformation of ring IV with respect to the rest of the tetrapyrrole ring plane of Chl(z)(.+) than Chl a(.+) probably induced by the protein matrix.