The rate of second electron transfer to Q B − in bacterial reaction center of impaired proton delivery shows hydrogen-isotope effect

Abstract

The 2nd electron transfer in reaction center of photosynthetic bacterium Rhodobacter sphaeroides is a two step process in which protonation of QB(-) precedes interquinone electron transfer. The thermal activation and pH dependence of the overall rate constants of different RC variants were measured and compared in solvents of water (H2O) and heavy water (D2O). The electron transfer variants where the electron transfer is rate limiting (wild type and M17DN, L210DN and H173EQ mutants) do not show solvent isotope effect and the significant decrease of the rate constant of the second electron transfer in these mutants is due to lowering the operational pKa of QB(-)/QBH: 4.5 (native), 3.9 (L210DN), 3.7 (M17DN) and 3.1 (H173EQ) at pH7. On the other hand, the proton transfer variants where the proton transfer is rate limiting demonstrate solvent isotope effect of pH-independent moderate magnitude (2.11±0.26 (WT+Ni(2+)), 2.16±0.35 (WT+Cd(2+)) and 2.34±0.44 (L210DN/M17DN)) or pH-dependent large magnitude (5.7 at pH4 (L213DN)). Upon deuteration, the free energy and the enthalpy of activation increase in all proton transfer variants by about 1 kcal/mol and the entropy of activation becomes negligible in L210DN/M17DN mutant. The results are interpreted as manifestation of equilibrium and kinetic solvent isotope effects and the structural, energetic and kinetic possibility of alternate proton delivery pathways are discussed.

Identifying involvement of Lys251/Asp252 pair in electron transfer and associated proton transfer at the quinone reduction site of Rhodobacter capsulatus cytochrome bc1

“…This could be result of another protonable group/groups, possibly water molecules, taking over the function of the original side chains that are missing in the mutants, or a reminiscence of natural existence of parallel (multiple) paths for protons in native protein [45]. The latter explanation is quite reasonable in light of the multiplicity for proton paths considered in the case of other quinone binding sites, such as the Q B site of photosynthetic reaction center [46], [47], [48]. However, the double mutants show that the simultaneous presence of non-protonable side chains at both positions (K251M/D252A, K251M/D252N) effectively deactivates proton entry to the Q i site which yields mutants non-functional in vivo with fully inactive Q i site.…”

Thermodynamic View of Proton Activated Electron Transfer in the Reaction Center of Photosynthetic Bacteria

“…The first H + ion is delivered directly to the C1–O group of Q B – from Asp–L213 through Ser–L223 and the second proton to Q B – from Asp–L213 via Glu–L212. From studies on different mutants, it was possible to estimate the Q – semiquinone of WT to have a p K a ≈ 4.5, quite similar to the value in aqueous solution. , …”

“…From studies on different mutants, it was possible to estimate the Q − semiquinone of WT to have a pK a ≈ 4.5, quite similar to the value in aqueous solution. 18,19 A series of former outstanding works 20−22 have provided a convincing description of the second electron transfer whose schematic energetic landscape is depicted in Figure 2.…”

“…As the protonation becomes the rate limiting step, the kinetics of the second electron transfer will show a deuterium isotope effect. 18 To understand this reaction fully, it is necessary to digest not just the kinetics but also the thermodynamics that reveals the energy levels, as well as the driving forces of the electron/ proton transfer. The driving force (the Gibbs free energy) of the reaction is composed of enthalpic and entropic components reflecting different components of the energetics of the reaction.…”

“…However, the roles are swapped in proton transfer mutants: the protonation is much slower than the subsequent electron transfer and controls the observed kinetics. As the protonation becomes the rate limiting step, the kinetics of the second electron transfer will show a deuterium isotope effect …”