CHARACTERIZATION OF THE CHLOROPLAST CYTOCHROME-B(6)F COMPLEX AS A STRUCTURAL AND FUNCTIONAL DIMER

Size analysis of the cytochrome b6f complex by FPLC Superose-12 chroma tography and Blue Native PAGE indicated a predominantly dimeric compon ent with M(r) = (1.9-2.5) X 10(5). The true dimer molecular weight inc luding bound lipid, but not detergent, was estimated to be 2.3 x 10(5) . Size and shape analysis by negative-stain single-particle electron m icroscopy indicated that the preparation of dimeric complexes contains a major population that has a protein cross section 40% larger than t he monomer, binds more negative stain, and has a geometry with a disti nct 2-fold axis of symmetry compared to the monomeric complex. The dim eric species is more stable at higher ionic strength with respect to c onversion to the monomeric species. SDS-PAGE of monomer and dimer prep arations indicated that both contain the four major polypeptides in ap proximately equal stoichiometry and also contain the petG M(r) 4000 su bunit. One bound chlorophyll a per monomer, part of the bound lipid, i s present in monomer and dimer. The in vitro electron-transport activi ty (decyl-PQH2 --> PC-ferricyanide) of the separated dimer was compara ble to that of the isolated b6f complex 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 mono mer was detected by SDS-PAGE or redox difference spectrophotometry tha t could account for the difference in activities. However, the concent ration of the Rieske [2Fe-2S] center was found by EPR analysis of the g(y) = 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 b6f complex, the absence of activity in th e monomer, and the absence of a monomer preparation that is not degrad ed in its spectral properties and activity suggest that the simplest i nference is that the dimer is the active complex in the membrane. The possibility that cytochrome b6f and bc1 are primitive trans-membrane-s ignaling complexes is noted.