Trapped conformational states of semiquinone (D+•QB-•) formed by B-Branch electron transfer at low temperature in Rhodobacter sphaeroides reaction centers

The reaction center ( RC) from Rhodobacter sphaeroides captures light energy by electron transfer between quinones Q(A) and Q(B), involving a conformational gating step. In this work, conformational states of D(+center dot)Q(B)(-center dot) were trapped (80 K) and studied using EPR spectroscopy in native and mutant RCs that lack QA in which QB was reduced by the bacteriopheophytin along the B-branch. In mutant RCs frozen in the dark, a light induced EPR signal due to D(+center dot)Q(B)(-center dot) formed in 30% of the sample with low quantum yield (0.2%-20%) and decayed in 6 s. A small signal with similar characteristics was also observed in native RCs. In contrast, the EPR signal due to D(+center dot)Q(B)(-center dot) in mutant RCs illuminated while freezing formed in similar to 95% of the sample did not decay (tau > 10(7) s) at 80 K ( also observed in the native RC). In all samples, the observed g-values were the same (g = 2.0026), indicating that all active Q(B)(-center dot)'s were located in a proximal conformation coupled with the nonheme Fe2+. We propose that before electron transfer at 80 K, the majority (similar to 70%) of Q(B), structurally located in the distal site, was not stably reducible, whereas the minority (similar to 30%) of active configurations was in the proximal site. The large difference in the lifetimes of the unrelaxed and relaxed D(+center dot)Q(B)(-center dot) states is attributed to the relaxation of protein residues and internal water molecules that stabilize D(+center dot)Q(B)(-center dot). These results demonstrate energetically significant conformational changes involved in stabilizing the D(+center dot)Q(B)(-center dot) state. The unrelaxed and relaxed states can be considered to be the initial and final states along the reaction coordinate for conformationally gated electron transfer.