Generation, characterization and crystallization of a cytochrome c1-subunit IV fused cytochrome bc1 complex from Rhodobacter sphaeroides

AbstractCytochrome bc1 complex catalyzes the reaction of electron transfer from ubiquinol to cytochrome c (or cytochrome c2) and couples this reaction to proton translocation across the membrane. Crystallization of the Rhodobacter sphaeroides bc1 complex resulted in crystals containing only three core subunits. To mitigate the problem of subunit IV being dissociated from the three-subunit core complex during crystallization, we recently engineered an R. sphaeroides mutant in which the N-terminus of subunit IV was fused to the C-terminus of cytochrome c1 with a 14-glycine linker between the two fusing subunits, and a 6-histidine tag at the C-terminus of subunit IV (c1-14Gly-IV-6His). The purified fusion mutant complex shows higher electron transfer activity, more structural stability, and less superoxide generation as compared to the wild-type enzyme. Preliminary crystallization attempts with this mutant complex yielded crystals containing four subunits and diffracting X-rays to 5.5Å resolution