Generation, Characterization And Crystallization Of A Subunit Iv Fused Mutant Cytochrome bc1 Complex From Rhodobacter Sphaeroides

Abstract

The cytochrome bc1 complex from Rhodobacter sphaeroides contains a three-subunit core complex and a supernumerary subunit (subunit IV). Although a 2.1 Å resolution x-ray crystallographic study of the wild-type complex has been achieved recently, the dissociation of subunit IV during crystallization has undermined structural information of subunit IV. To overcome this difficulty, we have constructed and characterized mutants with the N-terminus of subunit IV fused to the C-terminus of cyt. c1 (c1-IV fusion). A polyglycine (6 or 14 residues) linker was placed between the two involved proteins to ease the constraint that might result from the fusion of two subunits in the assembling process. A 6-histidine tag was placed at the C-terminus of IV (c1-6G-IVHis and c1-14G-IVHis) for the ease of purification. Both mutant cells grew photosynthetically at rates comparable to that of the wild-type cells. Although the bc1 complex can be purified from both mutants, the c1-14G-IVHis gave a better yield and higher activity. This purified fusion complex contains four protein subunits, has higher activity, and is more stable toward detergent treatment than the wild-type enzyme. Thus, it is suitable for the structure determination of the entire four-subunit complex. The x-ray crystallographic study of this fusion complex is in progress. This work was supported in part by a grant from NIH (GM30721).