LOCALIZATION OF THE 23-KDA SUBUNIT OF THE OXYGEN-EVOLVING COMPLEX OF PHOTOSYSTEM-II BY ELECTRON-MICROSCOPY

A dimeric photosystem II light-harvesting II super complex (PSII-LHCII SC), isolated by sucrose density gradient centrifugation, was previou sly structurally characterized [Borkema, E. J., Hankamer, B., Bald. D. , Kruip, J., Nield, J., Boonstra, A. F., Barber, J. & Rogner. M. (1995 ) Proc, Natl Alcan. Sci. USA 92, 175-179]. This PSII-LHCII SC bound th e 33-kDa subunit of the oxygen-evolving complex (OEC), but lacked the 33-kDa and 17-kDa subunits of the OEC. Herl the isolation procedure wa s modified by adding 1 M glycine betaine (1-carboxy-N,N,N-trimethylmet hanaminium hydroxide inner salt) to the sucrose gradient mixture. This procedure yielded PSII-LHCII SC that contained both the 33-kDa and th e 23-kDa subunits and had twice the oxygen-evolving capacity of the su per complexes lacking the 23-kDa polypeptide. Addition of CaCl2 to PSI I-LHCII SC with the 23-kDa subunit attached did not increase the oxyge n-evolution rate. This suggests that the 23-kDa subunit is bound in a functional manner and is present in significant amounts. Over 5000 par ticle projections extracted from electron microscope images of negativ ely stained PSII-LHCII SC, isolated in the presence and absence of gly cine betaine, were analyzed using single-particle image-averaging tech niques. Both the 33-kDa and 33-kDa subunits could be visualized in top -view and side-view projections. In the side view the 23-kDa subunit i s seen to protrude 0.5-1 nm further than the 33-kDa subunit, giving th e PSII particle a maximal height of 9.5 nm. Measured from the centres of the masses, the two 33-kDa subunits associated with the dimeric PSI I-LHCII SC are separated by 6.3 nm. The corresponding distance between the two 23-kDa subunits is 8.8 nm.