PURIFICATION AND CHARACTERIZATION OF THE CYTOCHROME B(6)F COMPLEX FROM CHLAMYDOMONAS-REINHARDTII

A protocol has been developed for the purification of the cytochrome b(6)f complex from the unicellular alga Chlamydomonas reinhardtii. It is based on the use of the neutral detergent Hecameg (6-O-(N-heptylcarbamoyl) methyl-alpha-D-glycopyranoside) and comprises only three steps: selective solubilization from thylakoid membranes, sucrose gradient sedimentation, and hydroxylapatite chromatography. The purified complex contains two b hemes (alpha bands, 564 nm; E(m,8) = -84 and -158 mV) and one chlorophyll alpha (lambda(max) = 667-668 nm) per cytochrome f (alpha band, 554 nm; E(m,8) = +330 mV). It is highly active in transferring electrons from decylplastoquinol to oxidized plastocyanin (turnover number 250-300 s(-1)). The purified complex contains seven subunits, whose identity has been established by N-terminal sequencing and/or peptide-specific immunolabeling, namely four high molecular weight subunits (cytochrome f, Rieske iron-sulfur protein, cytochrome b(6), and subunit IV) and three similar to 4-kDa miniproteins (PetG, PetL, and PetX). Stoichiometry measurements are consistent with every subunit being present as two copies per b(6)f dimer.