title: Purification and characterization of the cytochrome b6 f complex from Chlamydomonas reinhardtii.
abstract: A protocol has been developed for the purification of the cytochrome b6 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; Em,8 = -84 and -158 mV) and one chlorophyll alpha (lambda max = 667-668 nm) per cytochrome f (alpha band, 554 nm; Em,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 b6, and subunit IV) and three approximately 4-kDa miniproteins (PetG, PetL, and PetX). Stoichiometry measurements are consistent with every subunit being present as two copies per b6 f dimer.
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D016825:Chlamydomonas reinhardtii / Q000201:enzymology*; D003573:Cytochrome b Group / Q000032:analysis / Q000737:chemistry / Q000302:isolation & purification*; D045346:Cytochrome b6f Complex; D008969:Molecular Sequence Data; D010084:Oxidation-Reduction
chemical_list: D003573:Cytochrome b Group; D045346:Cytochrome b6f Complex