Iron-sulfur cluster reconstitution of spinach chloroplast Rieske protein requires a partially prefolded apoprotein.
The Rieske 2Fe-2S protein is a central component of the photosynthetic electron transport cytochrome b6f complex in chloroplast and cyanobacterial thylakoid membranes. We have constructed plasmids for expression in Escherichia coli of full-length and truncated Spinacia oleracea Rieske (PetC) proteins fused to the MalE, maltose binding protein. The expressed Rieske fusion proteins were found predominantly in soluble form in the E. coli cytoplasm. These proteins could be readily purified for further experimentation. In vitro reconstitution of the characteristic, "Rieske-type" 2Fe-2S cluster into these fused proteins was accomplished by a chemical method employing reduced iron and sulfide. Cluster incorporation was monitored by electron paramagnetic resonance and optical circular dichroism (CD) spectroscopy. CD spectral analysis in the ultraviolet region suggests that the spinach Rieske apoprotein must be in a partially folded conformation to incorporate an appropriate iron-sulfur cluster. These data further suggest that upon cluster integration, further folding occurs, allowing the Rieske protein to attain a final, native structure. The data presented here are the first to demonstrate successful chemical reconstitution of the 2Fe-2S cluster into a Rieske apoprotein from higher plant chloroplasts.
Gubernator
Beata
B
Institute of Biochemistry and Molecular Biology, University of Wroclaw, Przybyszewskiego 63/77, 51-148 Wroclaw, Poland.
Króliczewski
Jaroslaw
J
Kallas
Toivo
T
Szczepaniak
Andrzej
A
D001059:Apoproteins / Q000737:chemistry*; D002736:Chloroplasts / Q000737:chemistry; D002942:Circular Dichroism; D014450:Electron Transport Complex III / Q000096:biosynthesis / Q000737:chemistry*; D004926:Escherichia coli / Q000378:metabolism; D007506:Iron-Sulfur Proteins / Q000096:biosynthesis / Q000737:chemistry*; D017510:Protein Folding; D017433:Protein Structure, Secondary; D011993:Recombinant Fusion Proteins / Q000737:chemistry; D018724:Spinacia oleracea / Q000737:chemistry
D001059:Apoproteins; D007506:Iron-Sulfur Proteins; D011993:Recombinant Fusion Proteins; C037989:Rieske iron-sulfur protein; D014450:Electron Transport Complex III