Assembly of the Rieske iron-sulphur protein into the cytochrome bf complexin thylakoid membranes of isolated pea chloroplasts

The assembly of the Rieske iron-sulphur protein into the cytochrome bf comp lex was examined following import of S-35-labeled precursor protein by isol ated pea chloroplasts. Rieske protein assembled into the cytochrome bf comp lex was resolved from unassembled Rieske protein and from other membrane co mplexes by nondenaturing gel electrophoresis of dodecyl maltoside-solubiliz ed thylakoid membranes. Four mutant forms of the Rieske protein were able t o assemble into the cytochrome bf complex in isolated chloroplasts, These w ere a triple substitution mutant, C107S/H109R/C112S, replacing conserved re sidues involved in the ligation of the [2Fe-2S] centre; the mutant Delta 45 -52 which removed a glycine-rich region predicted to form a flexible hinge between the hydrophobic membrane-associated region and the hydrophilic lume nal domain; and mutants Delta 168-173 and Delta 177-179 which removed two C -terminal regions, which are highly conserved in chloroplast and cyanobacte rial Rieske proteins. This indicates that the [2Fe-2S] cluster, the glycine -rich region and the C-terminal region are not essential for stable assembl y of the Rieske protein into the cytochrome bf complex in isolated chloropl asts.