Manipulations of the B-Side Charge-Separated States' Energetics in the Rhodobacter sphaeroides Reaction Center

Abstract

The mutation HL(M182) in the Rb. sphaeroides reaction center (RC) results in the replacement of the monomer bacteriochlorophyll on the inactive side (B side) of the reaction center with a bacteriopheophytin (φB). In φB containing reaction centers, excitation of the initial electron donor, the special pair P, results in about 35% electron transfer along the normally inactive B side. However, the electron is transferred only to the exchanged cofactor φB. Several additional mutations in close proximity to bacteriopheophytins φB or HB have been created with the goal of altering the energetics of charge-separated states P+φB- and P+HB-. Aspartic acid residues were introduced to replace methionine L174 or valine M175 in the vicinity of the φB cofactor in order to raise the free energy of state P+φB-. Threonine M133 was mutated to the aspartic acid to add a hydrogen bond to the HB cofactor and lower the free energy of state P+HB-. The mutations in the environment surrounding the φB pigment resulted in a decrease in the quantum yield of P+φB- as well as a decrease in the recombination lifetime of this state. The mutation of valine M175 to aspartic acid showed the largest effect. The yield of state P+φB- decreased to about 25% and its recombination lifetime shortened from 200 to 125 ps. This additional mutation also resulted in the loss of the carotenoid molecule from the reaction centers. None of the three additional mutations altered the free energies sufficiently to result in observable electron transfer to HB. However, these measurements have allowed a more accurate assignment of the B-side charge-separated states' energetics than was previously possible.