Putative Hydrogen Bond to Tyrosine M208 in Photosynthetic Reaction Centers from Rhodobacter capsulatus Significantly Slows Primary Charge Separation

Abstract

Slow, ∼50 ps, P* → P+HA– electron transfer is observed in Rhodobacter capsulatus reaction centers (RCs) bearing the native Tyr residue at M208 and the single amino acid change of isoleucine at M204 to glutamic acid. The P* decay kinetics are unusually homogeneous (single exponential) at room temperature. Comparative solid-state NMR of [4′-13C]Tyr labeled wild-type and M204E RCs show that the chemical shift of Tyr M208 is significantly altered in the M204E mutant and in a manner consistent with formation of a hydrogen bond to the Tyr M208 hydroxyl group. Models based on RC crystal structure coordinates indicate that if such a hydrogen bond is formed between the Glu at M204 and the M208 Tyr hydroxyl group, the −OH would be oriented in a fashion expected (based on the calculations by Alden et al., J. Phys. Chem.1996, 100, 16761–16770) to destabilize P+BA– in free energy. Alteration of the environment of Tyr M208 and BA by Glu M204 via this putative hydrogen bond has a powerful influence on primary charge separation.

Switching sides—Reengineered primary charge separation in the bacterial photosynthetic reaction center

“…Complex kinetics were also found for P* → P + B A − → P + H A − and P + H A − → P + Q A − in WT RCs, implying multiple populations (4,37). P* populations that exhibit different photochemistry, such as seen here, also have been described for various mutants (9,33,44,62). These findings, the data here, and the model in Fig.…”

“…Both theory and experiments have shown that TyrM210 is key to positioning P + B A − slightly (∼70 meV) below P* in free energy, while symmetry-related PheL181 does not provide such stabilization of P + B B − , which is thought to be ∼100 to 200 meV above P* (Figs. 1 and 2A) (6)(7)(8)(9)(10)(11)(12)(13). State P + H B − also is higher in free energy than its A-side counterpart (P + H A − ) and may be only ∼70 meV below P* (14).…”

Genetic Code Expansion in Rhodobacter sphaeroides to Incorporate Noncanonical Amino Acids into Photosynthetic Reaction Centers

“…For instance, the tyrosine at M210 (Figure 1) has long been indicated as a key factor for L-branch electron transfer because it is a clear deviation in symmetry between the L and M branch protein environments, and it is the only 1 out of the 28 tyrosines in the RC whose hydroxyl group is not hydrogen-bonded. 6 Past simulation results have indicated that the orientation and dipole of the tyrosine hydroxyl play an important role in stabilizing initial charge transfer intermediates. 7 Recent results from our lab demonstrated the impact of introducing a putative hydrogen bond partner near Y(M210), a change which could reorient the tyrosine −OH dipole away from maximally stabilizing interactions for charge transfer indicated by these simulation results.…”

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

“…Despite the significant energetic impact of some ncAAs, no tyrosine modification made an order of magnitude impact on the overall rate (5,10,31,72) or yield (27) of ET, highlighting the robust nature of the RC's protein design (1,(4)(5)(6)(7)(8)(9)(10)(11). This small overall effect may be a consequence of this dualmechanism model, where the superexchange mechanism is acting as an alternative functional pathway for ET as previously proposed in the literature (19) through a lowered sensitivity to energetic changes of the P + BAintermediate.…”

“…Theoretical studies indicates that the magnitude and orientation of the hydroxyl dipole of tyrosine M210 may play an important role in energetically stabilizing P + BA - (29,30). Indeed, previous efforts to change the orientation of this tyrosine's hydroxyl dipole significantly slowed ET (31). It is difficult, however, to subtly vary the electrostatic nature of this tyrosine using canonical mutagenesis without entirely removing the phenolic hydroxyl.…”