Evidence for α-Tocopherol Function in the Electron Transport Chain of Chloroplasts

Abstract

' The effect of three different stable radicals-2,2-diphenyl-1-picrylhy-drazyl, 1,3,5-triphenyl-verdazyl, and galvinoxyl-was studied in photo-system II of spinach (Spinacia oleracea) chloroplasts. Inhibition by the three was noted on dimethylbenzoquinone reduction in presence of 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone (DBMIB) snd on sili-comolybdate reduction in presence of 3-(3,4-dichlorophenyl)-1,1-dime-thylurea (DCMU) in pbotosystem II and on the H20-) methylviologen reaction encompassing both photosystems. Inhibition of all photosystem II reactions except silicomolybdate reduction could be parilly restored by a-tocopherol or by 9-ethoxy-a-tocopherone but not by other qui-nones or radial chasers. On this basis, a functional role for a-tocopberol in the electron transport chain of spinach chloroplasts between the DCMU and DBMIB inhibition sites is postulated. Henninger et al. (12) proposed that tocopherols were involved in PSII of chloroplasts. This was based on the fact that chloro-plasts contain significant amounts of a-, ,3,-y-, and cr-tocoph-erol, that redox reactions occur as evidenced by the presence of the various tocopherol-quinone forms, and by the observation that (& and y-tocopherol could restore the photoreduction of ferricyanide in acetone-extracted chloroplasts. To the authors' knowledge, the acetone extraction results were never examined elsewhere. The only further development of ideas about tocoph-erol function in electron transport has been reported by Bishop and associates (6) who have a Scenedesmus mutant lacking a-tocopherol and which, therefore, is unable to function normally. There is evidence for restoring PSI activity in heptane-extracted chloroplasts by a-tocopherol, as shown by Baszynsky (4). Basz-ynski and Tukendorf (5) have also shown the reconstitution of cyclic photophosphorylation in heptane-extracted chloroplasts by a-tocopherol. Nishikimi and Machlin (18) showed that ca-tocopherol or 6-hydroxy-2,5,7,8-tetramethylchroman-2-carbox-ylic acid, a compound in which the isoprenoid side chain of a-tocopherol has been replaced by a carboxyl group, can be oxidized by the superoxide anion in a xanthine-xanthine oxidase system. We report a new approach to the function of a-tocopherol in PSII of spinach chloroplasts. It consists of the removal of to-copherol from the membrane with lipophilic free radicals and specific restoration of activity with a-tocopherol and 9-ethoxy-a-tocopherone. MATERIALS AND METHODS Spinach (Spinacia oleracea) chloroplasts were made from market spinach by a modified method of Jagendorf and Avron I