Isotope-based discrimination between the infrared modes of plastosemiquinone anion radicals and neutral tyrosyl radicals in photosystem II

Photosystem II (PSII) conducts the light-driven oxidation of water and redu ction of plastoquinone. Difference Fourier transform infrared (FT-IR) spect roscopy can be used to obtain information about structural changes which oc cur in protein and cofactors when charge separation occurs. The focus of th is work was the assignment of vibrational lines to two different species in PSII: the tyrosyl radical, Z ., and the plastosemiquinone anion radical, Q (A)(-) Difference FT-m experiments were conducted with cyanobacterial PSII samples, in which the tyrosine ring was uniformly C-13-labeled, in which ty rosine was C-13-labeled at carbon 4, and in which plastoquinone was methyl- deuterated. At 80 K, difference FT-LR spectra reflect the oxidation of chlo rophyll/carotenoid and the one-electron reduction of Q(A); no significant D or Z contribution to the spectrum is observed under these conditions. At 2 64 K, difference FT-IR spectra reflect the oxidation of redox-active tyrosi nes Z and D; no significant Q(A)(-) contribution is observed under these co nditions. At 80 K, isotope-induced shifts were observed in spectral feature s at 1482 and 1469 cm(-1) upon deuteration of plastoquinone. At 264 K, isot ope-induced shifts were observed in a 1478 cm(-1) line upon C-13- labeling of tyrosine, but little change was observed upon plastoquinone deuteration. These data support the assignment of a positive 1478 cm(-1) line to a tyro syl radical vibrational mode and positive 1482 and 1469 cm(-1) lines to pla stosemiquinone anion vibrational modes. Hybrid Hartree-Fock/density functio nal calculations of p-cresyl radical's vibrational frequencies and isotopic frequency shifts support this assignment.