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 + •Q B -• were trapped (80 K) and studied using EPR spectroscopy in native and mutant RCs that lack QA in which Q B was reduced by the bacteriopheophytin along the B-branch. In mutant RCs frozen in the dark, a light induced EPR signal due to D + •Q B -• 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 + •Q B -• in mutant RCs illuminated while freezing formed in ∼95% of the sample did not decay (T > 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 -•S were located in a proximal conformation coupled with the nonheme Fe 2+ . We propose that before electron transfer at 80 K, the majority (∼70%) of Q B , structurally located in the distal site, was not stably reducible, whereas the minority (∼30%) of active configurations was in the proximal site. The large difference in the lifetimes of the unrelaxed and relaxed D + •Q B -• states is attributed to the relaxation of protein residues and internal water molecules that stabilize D + •Q B -• These results demonstrate energetically significant conformational changes involved in stabilizing the D + •Q B -• 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.