Determination of the pigment stoichiometry of the photochemical reaction center of photosystem II

Abstract

The stoichiometry of chlorophyll a, pheophytin α and β-carotene in the photochemical reaction center of Photosystem II was analyzed by reversed phase high-performance liquid chromatography (HPLC) with methanol as the mobile phase, and by the shape of spectra of extracts in 80% acetone. For the HPLC method the molar extinction coefficient of pheophytin a in methanol was redetermined, while for the spectroscopic method spectra of extracts in 80% acetone were simulated by fitting with spectra of isolated chlorophyll a, pheophytin a and β-carotene in 80% acetone. Both methods give internally consistent results, and suggest that the reaction center of Photosystem II isolated by a short Triton X-100 treatment binds 6 chlorophyll a per 2 pheophytin a molecules. We also present evidence that prolonged exposure of the Photosystem II reaction center complex to Triton X-100 does not result in the loss of chlorophyll from the complex. Based on a comparison with spectra reported in publications from other groups, we conclude that the chlorophyll to pheophytin ratio has previously been underestimated to sometimes very significant extents, and that, as yet, no Photosystem II reaction center particles have been purified that bind less than 5–6 chlorophyll a per 2 pheophytin a.