Analysis of b₆f complex assembly in BN-PAGE and activity in vivo. (a) b₆f complexes after BN-PAGE of digitonin-solubilized membranes. Shown are a WT strain, petB-C35V (-c_i) strain, and petB-H202Q (-b_H) strain: a site-directed mutant of the chloroplast petB gene with a substitution of His202 involved as an axial ligand of b_H-heme preventing b_H-heme binding to cyt b₆. petB-H187G (-b_L) strain: site-directed mutant of the chloroplast petB with a substitution of His187 involved as axial ligand of b_L-heme preventing b_L-heme binding to cyt b₆. Digitonin-solubilized proteins were separated in the first dimension by BN-PAGE and in the second dimension by SDS-PAGE and analyzed by ECL immunodetection. b₆f complex migrates as a dimer of 320 kD (b₆f_dimer) and as a monomer of 140 kD (b₆f_monomer), both highlighted by arrows. (b) Fluorescence induction kinetics of dark-adapted cells from WT, petB-C35V, and ΔpetB strains.