Flash-induced changes in buffering capacity of reaction centers from photosynthetic bacteria reveal complex interaction between quinone pockets.
A novel method was applied to determine light-induced protonation in reaction centers from photosynthetic purple bacteria. Changes in buffering capacities upon flash excitation were detected in (0.03% Triton X-100) detergent solution of reaction centers from Rhodobacter (Rb.) sphaeroides and Rb. capsulatus wild type and mutant strains with empty or occupied secondary quinone (QB) binding sites in the presence of an external electron donor. The light-induced differences in buffering capacities between pH 4 and 11 were analyzed in terms of protonatable residues. Due to its differential nature, this method is more sensitive to the position and shift of pKa of the individual groups than the direct method based on proton uptake measurements. Out of the four different ionizable residues which were used to fit the curves, the two groups with apparent (dark) pKa values between 8.4-8.8 and 9.5-10.0 (depending on the species and conditions) disappeared when the native ubiquinone10 was replaced by menadione at the primary quinone (QA) binding site of Rb. sphaeroides or when the key protonatable residues (L212Glu and L213Asp) were replaced by non-protonatable alanines in the QB binding site of the AA+M43D mutant from Rb. capsulatus. The experimentally observed acidic and neutral residues remained unchanged. These results obtained from modifications in both quinone sites reveal the origin of the alkaline pH groups: they reflect the interaction of QA- and the cluster of ionizable residues around L212Glu in the QB binding pocket. The involvement of two residues with close pKa values reflects the complex titration of the cluster. The interaction between the quinone pockets is best described qualitatively as a network of ionizable residues extending from the QA site to the QB site.
Department of Biophysics, József Attila University, Egyetem utca 2, H-6722 Szeged, Hungary
L
Kálmán
P
Sebban
DK
Hanson
M
Schiffer
P
Maróti