Application of amber suppression to study the role of Tyr M210 in electron transfer in R. sphaeroides photosynthetic reaction centers

The initial light-induced electron transfer (ET) steps in the bacterial photosynthetic reaction center (RC) have been extensively studied and provide a paradigm for connecting structure and function. Although RCs have local pseudo-C2 symmetry, ET only occurs along the A branch of chromophores. Tyrosine M210 is a key symmetry-breaking residue adjacent to bacteriochlorophyll BA that bridges primary electron donor P and bacteriopheophytin acceptor HA. We used amber suppression to incorporate phenylalanine variants with different electron-withdrawing/donating capabilities at position M210. X-ray data generally reveal no appreciable structural changes due to the mutations. P* decay and P+HA formation are multi-exponential (~2-9, ~10-60, and ~100-300 ps) and temperature dependent. The 1020 nm transient-absorption band of P+BA is barely resolved for a few variants at 295 K and for none at 77 K. The results indicate a change from two-step ET for wild-type RCs to dominance of one-step superexchange ET for the mutants. Resonance Stark spectroscopy reveals that the free energy of P+BA changes by -57 to +66 meV among the phenylalanine variants. Because P+BA apparently lies above P* in all phenylalanine variants, the perturbations primarily affect the energy denominator for superexchange mixing. The findings deepen insight into primary ET in the bacterial RC.