Crosslinking of components of the cytochrome b6f complex from spinach thylakoids by o-phthalaldehyde.
Chemical modification of a purified spinach cytochrome (cyt) b6f complex was carried out using micromolar concentrations of o-phthalaldehyde (OPA). The o-phthalaldehyde-modified complex exhibited a strong and stable fluorescence characteristic of the thioisoindole derivative. o-Phthalaldehyde also caused crosslinking of the polypeptides and on polyacrylamide gel electrophoresis a new band with a molecular mass of 38-40 kDa exhibiting this characteristic fluorescence was observed as the major crosslinked component; in addition, minor protein bands in the mass range 50-60 kDa appeared. Immunostaining with specific antibodies identified the 38-kDa crosslink as a conjugate of cyt b6 with subunit IV and an additional conjugate of 54 kDa involving cyt f and subunit IV. Crosslinking of the Rieske protein with these components was not observed. In OPA-treated cyt b6f complex, the electron transfer from plastoquinol to cyt f is inhibited as well as proton translocation after incorporation of the complex into liposomes. The EPR spectral analysis of the complex modified at various o-phthalaldehyde concentrations showed that the characteristic EPR signal of the Rieske Fe-S center was lost.
Bhagwat
A S
AS
Technische Universität Berlin, Institut für Biochemie und Molekulare Biologie, Germany.
Blokesch
A
A
Irrgang
K D
KD
Salnikow
J
J
Vater
J
J
D002736:Chloroplasts / Q000737:chemistry* / Q000648:ultrastructure; D003432:Cross-Linking Reagents; D003573:Cytochrome b Group / Q000737:chemistry*; D045346:Cytochrome b6f Complex; D003580:Cytochromes / Q000737:chemistry*; D045348:Cytochromes f; D004579:Electron Transport / Q000187:drug effects; D007506:Iron-Sulfur Proteins / Q000737:chemistry; D046911:Macromolecular Substances; D010940:Plant Proteins / Q000737:chemistry; D010944:Plants; D009764:o-Phthalaldehyde / Q000737:chemistry* / Q000494:pharmacology
D003432:Cross-Linking Reagents; D003573:Cytochrome b Group; D003580:Cytochromes; D007506:Iron-Sulfur Proteins; D046911:Macromolecular Substances; D010940:Plant Proteins; D009764:o-Phthalaldehyde; D045346:Cytochrome b6f Complex; D045348:Cytochromes f