The IleL229 ? Met mutation impairs the quinone binding to the QB-pocket in reaction centers of Rhodobacter sphaeroides

Abstract

A spontaneous mutant (R/89) of photosynthetic purple bacterium Rhodobacter sphaeroides R-26 was selected for resistance to 200 μM atrazin. It showed increased resistance to interquinone electron transfer inhibitors of o-phenanthroline (resistance factor, RF=20) in UQo reconstituted isolated reaction centers and terbutryne in reaction centers (RF=55) and in chromatophores (RF=85). The amino acid sequence of the QB binding protein of the photosynthetic reaction center (the L subunit) was determined by sequencing the corresponding pufL gene and a single mutation was found (Ile(L229) → Met). The changed amino acid of the mutant strain is in van der Waals contact with the secondary quinone QB. The binding and redox properties of QB in the mutant were characterized by kinetic (charge recombination) and multiple turnover (cytochrome oxidation and semiquinone oscillation) assays of the reaction center. The free energy for stabilization of QAQB (-) with respect to QA (-)QB was ΔGAB=-60 meV and 0 meV in reaction centers and ΔGAB=-85 meV and -46 meV in chromatophores of R-26 and R/89 strains at pH 8, respectively. The dissociation constants of the quinone UQo and semiquinone UQo (-) in reaction centers from R-26 and R/89 showed significant and different pH dependence. The observed changes in binding and redox properties of quinones are interpreted in terms of differential effects (electrostatics and mesomerism) of mutation on the oxidized and reduced states of QB.

Light-Induced Equilibration Kinetics in Membrane-Bound Photosynthetic Reaction Centers: Nonlinear Dynamic Effects in Multiple Scattering Media

“…sphaeroides chromatophores that yielded a value of ∼85-120 meV for ∆G AB 0 . [25][26][27][28][29][30] The value of ∆G AB 0 is ∼60 meV for isolated Rb. sphaeroides RCs.…”

Structure and binding efficiency relations of QB site inhibitors of photosynthetic reaction centres*

“…sphaeroides R-26 cells were grown photoheterotrophically under anaerobic conditions in medium supplemented with potassium succinate (Ormerod et al 1961). Chromatophores and RCs were prepared as described earlier (Tandori et al 1995). RCs were solubilized by DDAO (N,N-dimethyldodecylamine N-oxide; Fluka) and purified by ammonium sulfate precipitation followed by DEAE-Sephacel (Sigma) anion-exchange chromatography.…”

“…Flash-induced absorption changes on the RC micelle and liposome samples (1.5 ml volume) were measured routinely by a single-beam kinetic spectrophotometer of local design (Tandori et al 1995). The P/P + redox changes of the bacteriochlorophyll dimer (primary donor of the photoinduced electron transfer, either in reduced P or oxidized P + form) were detected by monitoring the absorption change at 860 nm after flash excitation, during the back reaction (known also as charge recombination) from the oxidized P + to ground P state.…”

“…It is reasonable to hypothesize that the mode of action of the inhibitor depends essentially on the herbicide chemical structure and the protein environment, both influencing the nature of the interactions established at the binding site. The chemical structure of the compounds employed in our study and numbering of their atoms are summarized in Figs The herbicide efficiency on inhibiting the RC photochemistry, expressed in terms of binding free energy to the Q B site (∆G), and the relative order of inhibition ability were determined by measuring the recovery process from flash induced absorption changes at 860 nm in the presence of inhibitors (Wraight and Shopes 1989;Tandori et al 1995). Fig.…”

“…The fit allowed to determine the inhibition constant value (K I sometimes referred also as I 50 (Tandori et al 1995)) for each herbicide, defined as the herbicide concentration required to induce 50 % inhibition of the above mentioned Q B site activity.…”