Reorientation of the acetyl group of the photoactive bacteriopheophytin in reaction centers of Rhodobacter sphaeroides: An ENDOR/TRIPLE resonance study

The freeze-trapped bacteriopheophytin a radical anion Φ has been investigated by ¹H-ENDOR/Special TRIPLE resonance spectroscopy in photosynthetic reaction centers of Rhodobacter sphaeroides, in which the Tyr at position M210 had been replaced by either Phe, Leu, His or Trp. In the wild type reaction center and the mutants YF(M210) and YW(M210) two distinct states of Φ, denoted I and I, can be stabilized below 200 K. The state I is metastable and relaxes to I as the temperature is raised from 135 K to 180 K. The difference in the electronic structure of Φ between the two states is interpreted in terms of a conformational change of Φ_A after freeze-trapping, involving a reorientation of the 3-acetyl group with respect to the macrocycle of the bacteriopheophytin. This interpretation is supported by the results of RHF-INDO/SP calculations. In the YH(M210) reaction center only one Φ state is obtained that is distinct from I and I, and the observed electronic structure indicates an almost in-plane orientation of the 3-acetyl group. This is consistent with the proposal that a hydrogen bond is formed between His M210 and the 3¹-keto oxygen of Φ_A that impedes the reorientation of the acetyl group. Only one Φ state is observed in the YL(M210) reaction center, which is similar to the metastable state I in the wild type complex. This result is interpreted in terms of a steric hindrance of the reorientation of the 3-acetyl group that is exerted by the side chain of Leu at position M210. Possible implications of these findings for the mechanism of electron transfer in bacterial reaction centers are discussed. © 1999 John Wiley & Sons, Inc. Biospectroscopy 5: 35–46, 1999