Crosslinking of Components of the Cytochrome b6f Complex from Spinach Thylakoids by o-Phthalaldehyde

Abstract Chemical modification of a purified spinach cytochrome (cyt) b 6 f 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 b 6 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 b 6 f 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.