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.

B-Side Electron Transfer To Form P+HB- in Reaction Centers from the F(L181)Y/Y(M208)F Mutant of Rhodobacter capsulatus

“…In recent years this asymmetric functionality has been broken via site-directed mutagenesis producing RCs that undergo B-side electron transfer to give either P + H B -or P + φ B -(where φ B denotes a bacteriopheophytin that replaces B B ) in yields as high as 35%. [5][6][7][8][9][10][11][12][13] Charge-separated states on the B-side of the RC also have been reported under conditions of high pulse energy or short wavelength excitation with native complexes. 14,15 Recently it was shown that P + H B -supports electron transfer to Q B , with the rate more than 10-fold slower than P + H A -f P + Q A -electron transfer.…”

Species differences in unlocking B‐side electron transfer in bacterial reaction centers

“…B), which mirrors the A‐side residue L104Glu known to form a hydrogen bond with H A . Previous work has shown that Asp or Glu substituted at M133/M131 can form a hydrogen bond with H B , promoting its reduction. The combination of the V(M131)E (‘E’) substitution with the ‘core’ FHV and YFHV mutations in the R. capsulatus RC has resulted in large increases in yields of B‐side ET .…”

Temperature Dependence of Electron Transfer to the M-Side Bacteriopheophytin in Rhodobacter capsulatus Reaction Centers

“…[55][56][57] Additionally, calculations generally place the P + B -state higher in free energy than the P + H -state on both L and M branches by 0.15-0.25 eV, consistent with the difference in redox potentials of bacteriochlorophyll and bacteriopheophytin in vitro. [58][59][60][61] Calculations and experiments have provided estimates or bracketed ranges for the free energies of the charge-separated states in the wild-type RC: P + B L -0.05-0.1 eV below P*; 29,50,51,[62][63][64][65][66][67][68][69][70] P + H L -∼0.25 eV below P* when relaxed; [71][72][73][74][75] P + B M -0.1-0.2 eV above P* and P + H M -below P* by no more than ∼0.15 eV and probably within 0.1 eV. [50][51][52][76][77][78][79] Systematic efforts to manipulate the free energy differences of the L-and M-branch charge-separated states by site-directed mutagenesis of key amino acids, including those near B M and B L , have led to mutant RCs in which electron transfer to the M branch competes effectively with charge separation to the L branch, yielding P + H M -(reviewed in ref 80).…”