Mutational control of bioenergetics of bacterial reaction center probed by delayed fluorescence

The free energy gap between the metastable charge separated state P⁺Q_A^? and the excited bacteriochlorophyll dimer P* was measured by delayed fluorescence of the dimer in mutant reaction center proteins of the photosynthetic bacterium Rhodobacter sphaeroides. The mutations were engineered both at the donor (L131L, M160L, M197F and M202H) and acceptor (M265I and M234E) sides. While the donor side mutations changed systematically the number of H-bonds to P, the acceptor side mutations modified the energetics of Q_A by altering the van-der-Waals and electronic interactions (M265IT) and H-bond network to the acidic cluster around Q_B (M234EH, M234EL, M234EA and M234ER). All mutants decreased the free energy gap of the wild type RC (~ 890 meV), i.e. destabilized the P⁺Q_A^? charge pair by 60–110 meV at pH 8. Multiple modifications in the hydrogen bonding pattern to P resulted in systematic changes of the free energy gap. The destabilization showed no pH-dependence (M234 mutants) or slight increase (WT, donor-side mutants and M265IT above pH 8) with average slope of 10–15 meV/pH unit over the 6–10.5 pH range. In wild type and donor-side mutants, the free energy change of the charge separation consisted of mainly enthalpic term but the acceptor side mutants showed increased entropic (even above that of enthalpic) contributions. This could include softening the structure of the iron ligand (M234EH) and the Q_A binding pocket (M265IT) and/or increase of the multiplicity of the electron transfer of charge separation in the acceptor side upon mutation.