Infrared spectroscopic identification of the C-O stretching vibration associated with the tyrosyl Z. and D. radicals In photosystem II
Photosystem II (PSII) is a multisubunit complex, which catalyzes the photo-induced oxidation of water and reduction of plastoquinone. Difference Fourier-transform infrared (FT-IR) spectroscopy can be used to obtain information about the structural changes accompanying oxidation of the redox-active tyrosines, D and Z, in PSII. The focus of our work is the assignment of the 1478 cm-1 vibration, which is observable in difference infrared spectra associated with these tyrosyl radicals. The first set of FT-IR experiments is performed with continuous illumination. Use of cyanobacterial strains, in which isotopomers of tyrosine have been incorporated, supports the assignment of a positive 1478/1477 cm-1 mode to the C-O stretching vibration of the tyrosyl radicals. In negative controls, the intensity of this spectral feature decreases. The negative controls involve the use of inhibitors or site-directed mutants, in which the oxidation of Z or D is eliminated, respectively. The assignment of the 1478/1477 cm-1 mode is also based on control EPR and fluorescence measurements, which demonstrate that no detectable Fe+2QA- signal is generated under FT-IR experimental conditions. Additionally, the difference infrared spectrum, associated with formation of the S2QA- state, argues against the assignment of the positive 1478 cm-1 line to the C-O vibration of QA-. In the second set of FT-IR experiments, single turnover flashes are employed, and infrared difference spectra are recorded as a function of time after photoexcitation. Comparison to kinetic transients generated in control EPR experiments shows that the decay of the 1477 cm-1 line precisely parallels the decay of the D. EPR signal. Taken together, these two experimental approaches strongly support the assignment of a component of the 1478/1477 cm-1 vibrational lines to the C-O stretching modes of tyrosyl radicals in PSII. Possible reasons for the apparently contradictory results of Hienerwadel et al. (1996) Biochemistry 35, 15,447-15,460 and Hienerwadel et al. (1997) Biochemistry 36, 14,705-14,711 are discussed. Copyright 1998 Elsevier Science B.V. All rights reserved.
Kim
S
University of Minnesota, College of Biological Sciences, Department of Biochemistry, St. Paul, MN 55108, USA.
Ayala
I
Steenhuis
JJ
Gonzalez
ET
Barry
BA