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

The assembly of the Rieske iron–sulphur protein into the cytochrome bf complex was examined following import of 35S‐labeled precursor protein by isolated pea chloroplasts. Rieske protein assembled into the cytochrome bf complex was resolved from unassembled Rieske protein and from other membrane complexes by nondenaturing gel electrophoresis of dodecyl maltoside‐solubilized thylakoid membranes. Four mutant forms of the Rieske protein were able to assemble into the cytochrome bf complex in isolated chloroplasts. These were a triple substitution mutant, C107S/H109R/C112S, replacing conserved residues involved in the ligation of the [2Fe‐2S] centre; the mutant Δ45–52 which removed a glycine‐rich region predicted to form a flexible hinge between the hydrophobic membrane‐associated region and the hydrophilic lumenal domain; and mutants Δ168–173 and Δ177–179 which removed two C‐terminal regions, which are highly conserved in chloroplast and cyanobacterial Rieske proteins. This indicates that the [2Fe–2S] cluster, the glycine‐rich region and the C‐terminal region are not essential for stable assembly of the Rieske protein into the cytochrome bf complex in isolated chloroplasts.