Application of Amber Suppression To Study the Role of Tyr M210 in Electron Transfer in Rhodobacter sphaeroides Photosynthetic Reaction Centers.

Abstract

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 B_A that bridges the primary electron donor P and the bacteriopheophytin acceptor H_A. We used amber suppression to incorporate phenylalanine variants with different electron-withdrawing/-donating capabilities at the position M210. X-ray data generally reveal no appreciable structural changes due to the mutations. P* decay and P⁺H_A^- formation are multiexponential (∼2 to 9, ∼10 to 60, and ∼100 to 300 ps) and temperature dependent. The 1020 nm transient-absorption band of P⁺B_A^- 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 the dominance of one-step superexchange ET for the mutants. Resonance Stark spectroscopy reveals that the free energy of P⁺B_A^- changes by -57 to +66 meV among the phenylalanine variants. Because P⁺B_A^- 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.