REQUIREMENT OF HISTIDINE-217 FOR UBIQUINONE REDUCTASE-ACTIVITY (Q(I)-SITE) IN THE CYTOCHROME-BC(1) COMPLEX

Folding models suggest that the highly conserved histidine 217 of the cytochrome b subunit from the cytochrome bc1 complex is close to the quinone reductase (Q(i)) site. This histidine (bH217) in the cytochrome b polypeptide of the photosynthetic bacterium Rhodobacter capsulatus has been replaced with three other residues, aspartate (D), arginine (R), and leucine (L). bH217D and bH217R are able to grow photoheterotrophically and contain active cytochrome bc1 complexes (60% of wild-type activity), whereas the bH217L mutant is photosynthetically incompetent and contains a cytochrome bc1 complex that has only 10% of the wild-type activity. Single-turnover flash-activated electron transfer experiments show that cytochrome b(H) is reduced via the Q(o) site with near native rates in the mutant strains but that electron transfer between cytochrome b(H) and quinone bound at the Q(i) site is greatly slowed. These results are consistent with redox midpoint potential (E(m)) measurements of the cytochrome b subunit hemes and the Q(i) site quinone. The E(m) values of cyt b(L) and b(H) are approximately the same in the mutants and wild type, although the mutant strains have a larger relative concentration of what may be the high-potential form of cytochrome b(H), called cytochrome b150. However, the redox properties of the semiquinone at the Q(i) site are altered significantly. The Q(i) site semiquinone stability constant of bH217R is 10 times higher than in the wild type, while in the other two strains (bH217D and bH217L) the stability constant is much lower than in the wild type. Thus H217 appears to have major effects on the redox properties of the quinone bound at the Q(i) site. These data are incorporated into a suggestion that H217 forms part of the binding pocket of the Q(i) site in a manner reminiscent of the interaction between quinone bound at the Q(b) site and H190 of the L subunit of the bacterial photosynthetic reaction center.