HISTIDINE OXIDATION IN THE S-2 TO S-3 TRANSITION PROBED BY FTIR DIFFERENCE SPECTROSCOPY IN THE CA2+-DEPLETED PHOTOSYSTEM-II - COMPARISON WITH HISTIDINE RADICALS GENERATED BY UV IRRADIATION

FTIR difference and EPR spectroscopies were used to identify the organic radical species formed during the S-2 to S-3 transition in Ca2+-depleted, EGTA-treated, and polypeptide-reconstituted photosystem II membranes (denoted S-2' and S-3', respectively). Ferricyanide was added to the samples to act as an exogenous electron acceptor. Using EPR spectroscopy, it was shown that, under the experimental conditions used, only the species oxidized in the S-3' State was detected during the time required for the acquisition of the FTIR difference spectra. No contributions from the electron acceptor side were observed. The corresponding S-3'/S-2' FTIR difference spectra were recorded at 10 degrees C in H2O, D2O, and with N-15-labeled photosystem II membranes. Spectra were compared with radical-minus-neutral FTIR difference spectra of amino acid model compounds generated by UV irradiation at low temperature. Under our experimental conditions, we did not observe FTIR difference Signals consistent with tyrosine oxidation in the S-2' to S-3' transition. The infrared signals characteristic of radical formation with 4-methylimidazole and histidine obtained by UV irradiation of 4-methylimidazolium at pH 6 and of a His-Tyr dipeptide at pH 7 are presented. The analogy found between these spectra and the S-3'/S-2' spectrum obtained in situ supports the oxidation of a histidinium in the S-2' to S-3' transition.