Characterization of the Chloroplast Cytochrome b6f Complex as a Structural and Functional Dimer

Size analysis of the cytochrome bdcomplex by FPLC Superose-12 chromatography and Blue Native PAGE indicated a predominantly dimeric component with M, = (1.9-2.5) X lo5. The true dimer molecular weight including bound lipid, but not detergent, was estimated to be 2.3 X lo5. Size and shape analysis by negative-stain single-particle electron microscopy indicated that the preparation of dimeric complexes contains a major population that has a protein cross section 40% larger than the monomer, binds more negative stain, and has a geometry with a distinct 2-fold axis of symmetry compared to the monomeric complex. The dimeric species is more stable at higher ionic strength with respect to conversion to the monomeric species. SDS-PAGE of monomer and dimer preparations indicated that both contain the four major polypeptides in approximately equal stoichiometry and also contain thepetG M, 4000 subunit. One bound chlorophyll a per monomer, part of the bound lipid, is present in monomer and dimer. The in vitro electron-transport activity (decyl-PQHz - PC-ferricyanide) of the separated dimer was comparable to that of the isolated bdcomplex and was 4-5-fold greater than that of the monomer preparation, whose activity could be attributed to residual dimer. No difference in the properties of the dimer and monomer was detected by SDS-PAGE or redox difference spectrophotometry that could account for the difference in activities. However, the concentration of the Rieske (2Fe-2S) center was found by EPR analysis of the g, = 1.90 signal to be lower in the monomer fraction by a factor of 3.5 relative to the dimer. The presence of active dimer at high levels in the detergent-extracted bdcomplex, the absence of activity in the monomer, and the absence of a monomer preparation that is not degraded in its spectral properties and activity suggest that the simplest inference is that the dimer is the active complex in the membrane. The possibility that cytochrome bd and bcl are primitive trans-membrane-signaling complexes is noted.