Characterization of the chloroplast cytochrome b6f complex as a structural and functional dimer.
Size analysis of the cytochrome b6f complex by FPLC Superose-12 chromatography and Blue Native PAGE indicated a predominantly dimeric component with M(r) = (1.9-2.5) x 10(5). The true dimer molecular weight including bound lipid, but not detergent, was estimated to be 2.3 x 10(5). 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 the petG M(r) 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-PQH2-->PC-ferricyanide) of the separated dimer was comparable 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 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 gy = 1.90 signal to be lower in the monomer fraction by a factor of 3.5 relative to the dimer.(ABSTRACT TRUNCATED AT 250 WORDS)
Huang
D
D
Department of Biological Sciences, Purdue University, West Lafayette, Indiana 47907.
Everly
R M
RM
Cheng
R H
RH
Heymann
J B
JB
Schägger
H
H
Sled
V
V
Ohnishi
T
T
Baker
T S
TS
Cramer
W A
WA
D001704:Biopolymers; D002736:Chloroplasts / Q000201:enzymology*; D002850:Chromatography, Gel; D003573:Cytochrome b Group / Q000737:chemistry / Q000378:metabolism* / Q000648:ultrastructure; D045346:Cytochrome b6f Complex; D014450:Electron Transport Complex III; D004591:Electrophoresis, Polyacrylamide Gel; D007506:Iron-Sulfur Proteins / Q000737:chemistry; D008970:Molecular Weight; D010084:Oxidation-Reduction; D011487:Protein Conformation; D013057:Spectrum Analysis
D001704:Biopolymers; D003573:Cytochrome b Group; D007506:Iron-Sulfur Proteins; C037989:Rieske iron-sulfur protein; D045346:Cytochrome b6f Complex; D014450:Electron Transport Complex III