Photosynthetic reaction center variants made via genetic code expansion show Tyr at M210 tunes the initial electron transfer mechanism.

Abstract

Photosynthetic reaction centers (RCs) from <i>Rhodobacter sphaeroides</i> were engineered to vary the electronic properties of a key tyrosine (M210) close to an essential electron transfer component via its replacement with site-specific, genetically encoded noncanonical amino acid tyrosine analogs. High fidelity of noncanonical amino acid incorporation was verified with mass spectrometry and X-ray crystallography and demonstrated that RC variants exhibit no significant structural alterations relative to wild type (WT). Ultrafast transient absorption spectroscopy indicates the excited primary electron donor, P*, decays via a ∼4-ps and a ∼20-ps population to produce the charge-separated state P⁺H_A^- in all variants. Global analysis indicates that in the ∼4-ps population, P⁺H_A^- forms through a two-step process, P*→ P⁺B_A^-→ P⁺H_A^-, while in the ∼20-ps population, it forms via a one-step P* → P⁺H_A^- superexchange mechanism. The percentage of the P* population that decays via the superexchange route varies from ∼25 to ∼45% among variants, while in WT, this percentage is ∼15%. Increases in the P* population that decays via superexchange correlate with increases in the free energy of the P⁺B_A^- intermediate caused by a given M210 tyrosine analog. This was experimentally estimated through resonance Stark spectroscopy, redox titrations, and near-infrared absorption measurements. As the most energetically perturbative variant, 3-nitrotyrosine at M210 creates an ∼110-meV increase in the free energy of P⁺B_A^- along with a dramatic diminution of the 1,030-nm transient absorption band indicative of P⁺B_A^- formation. Collectively, this work indicates the tyrosine at M210 tunes the mechanism of primary electron transfer in the RC.