DIFFERENCE FT-IR STUDY OF A NOVEL BIOCHEMICAL PREPARATION OF PHOTOSYSTEM-II

There are two redox-active tyrosines in photosystem II, the water-splitting complex, that form neutral tyrosine radicals. One of these tyrosine radicals, D., is stable and has an unknown function. The other redox-active tyrosine, Z, acts to transfer oxidizing equivalents from the primary chlorophyll donor of photosystem II to the manganese cluster, where water oxidation occurs. In an attempt to obtain more information about Z and its interaction with its environment, we have begun a study using Fourier-transform infrared (FT-IR) vibrational spectroscopy. To facilitate these studies, we have developed a procedure to isolate spinach photosystem II complexes with an antenna size of approximately 100-110 chlorophylls per reaction center. These complexes show an approximately 2-fold increase in the specific activity of oxygen evolution over the activity of the starting material, photosystem II membranes. Although fully solubilized in detergent, these complexes retain the 24- and 18-kDa extrinsic proteins and exhibit no calcium chloride requirement for optimal oxygen evolution. In manganese-depleted photosystem II samples, Z. can be accumulated in the light. In the dark, the tyrosine radical is reduced and reprotonated to form the neutral tyrosine. Since this process is reversible and light-dependent, we have used light-minus-dark difference FT-IR spectroscopy to observe the vibrational difference spectrum that is associated with the oxidation of this residue. As a control, EPR spectra were measured under identical conditions to assess the amount of Z. that accumulated in the light. We also hope to use difference FT-IR to identify the amino acid with which Z may form a hydrogen bond. As a first step, we have obtained the difference FT-IR spectrum associated with protonation of the imidazole ring of histidine in vitro. This spectrum allows us to assess the contribution that such a protonation would have on the in vivo difference spectrum.