Conformational relaxation following reduction of the photoactive bacteriopheophytin in reaction centers from Blastochloris viridis. Influence of mutations at position M208

The photochemically trapped bacteriopheophytin (BPh) b radical anion in the active branch (Phi(A)(.-)) of reaction centers (RCs) from Blastochloris (f ormerly called Rhodopseudomonas) viridis is characterized by H-1-ENDOR as w ell as optical absorption spectroscopy. The two site-directed mutants YF(M2 08) and YL(M208), in which tyrosine at position M208 is replaced by phenyla lanine and leucine, respectively, are investigated and compared with the wi ld type. The residue at M208 is in close proximity to the primary electron donor, P, the monomeric bacteriochlorophyll (BChl), B-A, and the BPh, Phi(A ), that are involved in the transmembrane electron transfer to the quinone, Q(A), in the RC. The analysis of the ENDOR spectra of Phi(A)(.-) at 160 K indicates that two distinct states of Phi(A)(.-) are present in the wild ty pe and the mutant YF(M208), Based on a comparison with Phi(.)(A-) in RCs of Rhodobacter sphaeroides the two states are interpreted as torsional isomer s of the 3-acetyl group of Phi(A). Only one Phi(A)(.) state occurs in the m utant YL(M208). This effect of the leucine residue at position M208 is expl ained by steric hindrance that locks the acetyl group in one specific posit ion. On the basis of these results, an interpretation of the optical absorp tion difference spectrum of the state Phi(A)(.-)Phi(A)(.-) is attempted. Th is state can be accumulated at 100 K and undergoes an irreversible change b etween 100 and 200 K [Tiede et al., Biochim. Biophys. Acta 892 (1987) 294-3 02]. The corresponding absorbance changes in the BChl Q(x) and Q(y) regions observed in the wild type also occur in the YF(M208) mutant but not in YL( M208). The observed changes in the wild type and YF(M208) are assigned to R Cs in which the 3-acetyl group of Phi(A) changes its orientation. It is con cluded that this distinct structural relaxation of Phi(A) can significantly affect the optical properties of BA and contribute to the light-induced ab sorption difference spectra. (C) 2000 Elsevier Science B.V. All rights rese rved.