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

Abstract

The bacterial photosynthetic reaction center (RC) is an important system to understand the structure and chemistry of the biological process of photosynthesis. In wild type reaction centers and at room temperature, the unidirectional electron transfer from the excited-primary donor P∗ to the A-side bacteriopheophytin HA takes place via a two-step mechanism, where P∗ transfers an electron to the A-side bacteriochlorphyll BA in 3 ps making P+BA-, which then transfers an electron in 1 ps to HA to make P+HA-. Even though RCs have a pseudo-C2 symmetry, electron transfer only occurs along the A-branch. Tyrosine M210 has been a key amino acid of particular interest since it is a clear deviation in symmetry between the two sides of RC, and many mutations have been made at this site. One interesting finding was that when M210 is mutated to phenylalanine, the electron transfer from P∗ and HA slows down from ∼4 ps to ∼11 ps and becomes temperature independent (Nagarajan et al. 1990, PNAS). By using amber suppression, we were able to incorporate phenylalanine variants with different electron-withdrawing/donating capabilities at position M210. To our surprise, transient absorption data shows that YM210F RCs do not exhibit temperature independent primary charge separation. In fact, YM210F RCs and all M210 phenylalanine variants RCs exhibit Arrhenius temperature dependence in contrast to WT, wherein the rate increases at low temperature. Significantly, the 1020 nm band, which is characteristic of P+BA-, was not observed at any time for any of the M210 phenylalanine variants RCs nor YM210F RCs, indicating a change to the one-step superexchange mechanism for electron transfer.