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 previously structurally characterized [Boekema, E. J., Hankamer, B., Bald, D., Kruip, J., Nield, J., Boonstra, A. F., Barber, J. & Rögner, M. (1995) Proc. Natl Acad. Sci. USA 92, 175-179]. This PSII-LHCII SC bound the 33-kDa subunit of the oxygen-evolving complex (OEC), but lacked the 23-kDa and 17-kDa subunits of the OEC. Here the isolation procedure was modified by adding 1 M glycine betaine (1-carboxy-N,N,N-trimethylmethanaminium hydroxide inner salt) to the sucrose gradient mixture. This procedure yielded PSII-LHCII SC that contained both the 33-kDa and the 23-kDa subunits and had twice the oxygen-evolving capacity of the super complexes lacking the 23-kDa polypeptide. Addition of CaCl2 to PSII-LHCII SC with the 23-kDa subunit attached did not increase the oxygen-evolution rate. This suggests that the 23-kDa subunit is bound in a functional manner and is present in significant amounts. Over 5000 particle projections extracted from electron microscope images of negatively stained PSII-LHCII SC, isolated in the presence and absence of glycine betaine, were analyzed using single-particle image-averaging techniques. Both the 23-kDa and 33-kDa subunits could be visualized in top-view and side-view projections. In the side view the 23-kDa subunit is seen to protrude 0.5-1 nm further than the 33-kDa subunit, giving the 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 PSII-LHCII SC are separated by 6.3 nm. The corresponding distance between the two 23-kDa subunits is 8.8 nm.
Boekema
E J
EJ
Biophysical Chemistry, Groningen Biomolecular Sciences and Biotechnology Institute, University of Groningen, The Netherlands. boekema@chem.rug.nl
Nield
J
J
Hankamer
B
B
Barber
J
J
D001622:Betaine; D007091:Image Processing, Computer-Assisted; D008854:Microscopy, Electron; D009942:Organometallic Compounds / Q000378:metabolism; D010100:Oxygen / Q000378:metabolism; D045322:Photosynthetic Reaction Center Complex Proteins / Q000737:chemistry* / Q000648:ultrastructure; D045332:Photosystem II Protein Complex; D010940:Plant Proteins / Q000737:chemistry / Q000302:isolation & purification; D018724:Spinacia oleracea / Q000648:ultrastructure
D009942:Organometallic Compounds; D045322:Photosynthetic Reaction Center Complex Proteins; D045332:Photosystem II Protein Complex; D010940:Plant Proteins; C064388:oxygen-evolving complex, 33 kDa protein; C005460:uranyl acetate; D001622:Betaine; D010100:Oxygen