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

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 (φ_B). In φ_B 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 φ_B. Several additional mutations in close proximity to bacteriopheophytins φ_B or H_B have been created with the goal of altering the energetics of charge-separated states P⁺φ_B^- and P⁺H _B^-. Aspartic acid residues were introduced to replace methionine L174 or valine M175 in the vicinity of the φ_B cofactor in order to raise the free energy of state P^-φ _B^-. Threonine M133 was mutated to the aspartic acid to add a hydrogen bond to the H_B cofactor and lower the free energy of state P⁺H_B^-. The mutations in the environment surrounding the φ_B pigment resulted in a decrease in the quantum yield of P⁺φ_B^- 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 ⁺φ_B^- 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 H_B. However, these measurements have allowed a more accurate assignment of the B-side charge-separated states' energetics than was previously possible.