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Articles

Extraction and Reconstitution of Photosystem II

^a Department of Biology, Revelle College, University of California, San Diego, La Jolla, California 92037

Hill activity (oxygen evolution with ferricyanide as the electron acceptor), light-induced absorbance changes at liquid nitrogen temperature associated with the primary activity of photosystem II, and fluorescence yield changes at both low temperature and room temperature were measured with lyophilized spinach chloroplasts before and after extraction with hexane and reconstitution with -carotene and plastoquinone A. Extraction eliminated the Hill activity, and both -carotene and plastoquinone A were required for maximal restoration of activity to the reconstituted chloroplasts.

Extraction also eliminated the light-induced absorbance changes at –196 C due to the photoreduction of C-550 and photooxidation of cytochrome b₅₅₉, and reconstitution with -carotene and plastoquinone A restored the low temperature photoreactions. However, only -carotene was essential for the restoration of the photoreactions. Cytochrome b₅₅₉ was modified, as a result of the extraction, to a lower redox potential, autooxidizable form and remained as such after reconstitution with -carotene. The -carotene-restored chloroplasts showed the photoreduction of C-550 but not the photooxidation of cytochrome b₅₅₉ because the cytochrome was already oxidized. When -carotene-reconstituted chloroplasts were suspended in buffer containing ascorbate prior to freezing, the cytochrome b₅₅₉ was reduced and could be photooxidized by irradiation at low temperature. After reconstitution with -carotene plus plastoquinone A the cytochrome b₅₅₉ was partially restored to its original high potential form and was in the reduced state so that both the photoreduction of C-550 and the photooxidation of cytochrome b₅₅₉ occurred on irradiation of the -carotene plus plastoquinone A-reconstituted chloroplasts. Reconstitution with plastoquinone A alone had essentially no effect on restoring the photoreactions.

The fluorescence yield of dark-adapted lyophilized chloroplasts at –196 C showed an irreversible increase of about 2.5-fold during irradiation. After extraction the fluorescence yield of the chloroplasts was high (at the maximal light-induced level of the lyophilized control chloroplasts) and showed very little change in the light. Reconstitution with -carotene alone restored some fluorescence quenching which was relieved by irradiation at low temperature. Reconstitution with plastoquinone A alone restored a high degree of quenching, but this quenching was not relieved by light at low temperature. Fluorescence emission spectra at –196 C showed that the fluorescence of variable yield in the lyophilized and -carotene-reconstituted chloroplasts involved only the 680 and 695 nm emission bands but not the larger 730 nm emission band, whereas the irreversible quenching in plastoquinone A-reconstituted chloroplasts involved all wavelengths of emission. Extraction of the chloroplasts also eliminated the sharp 695 nm emission band at low temperature, and reconstitution with -carotene partially restored it.

The fluorescence yield changes at room temperature differed from the low temperature measurements in that the strong fluorescence quenching restored to the plastoquinone A-reconstituted chloroplasts was relieved by light and reappeared in the dark. Thus plastoquinone A appeared to be much more effective than -carotene in restoring the fluorescence of variable yield in room temperature measurements. However, it is argued from the results at low temperature that the quenching in plastoquinone A-reconstituted chloroplasts, which is probably due to the oxidized form of the quinone, is nonspecific and a different quenching mechanism from that which obtains in normal chloroplasts.

The results suggest that extraction with hexane removes plastoquinone A, which interrupts electron transport, and -carotene, which disrupts the primary photochemical activity of photosystem II. Reconstitution of the extracted chloroplasts with -carotene alone restores C-550 and the primary photochemical activity of photosystem II, and when the photosystem II reaction centers are restored the additional requirement of plastoquinone A for the Hill reaction can be demonstrated.

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