SITE-SPECIFIC AND COMPENSATORY MUTATIONS IMPLY UNEXPECTED PATHWAYS FOR PROTON DELIVERY TO THE Q(B) BINDING-SITE OF THE PHOTOSYNTHETIC REACTION-CENTER

In photosynthetic reaction centers, a quinone molecule, Q(B), is the t erminal acceptor in light-induced electron transfer. The protonatable residues Glu-L212 and Asp-L213 have been implicated in the binding of Q(B) and in proton transfer to Q(B) anions generated by electron trans fer from the primary quinone Q(A). Here we report the details of the c onstruction of the Ala-L212/Ala-L213 double mutant strain by site-spec ific mutagenesis and show that its photosynthetic incompetence is due to an inability to deliver protons to the Q(B) anions. We also report the isolation and biophysical characterization of a collection of reve rtant and suppressor strains that have regained the photosynthetic phe notype. The compensatory mutations that restore function are diverse a nd show that neither Glu-L212 nor Asp-L213 is essential for efficient light-induced electron or proton transfer in Rhodobacter capsulatus. S econd-site mutations, located within the Q(B) binding pocket or at mor e distant sites, can compensate for mutations at L212 and L213 to rest ore photocompetence. Acquisition of a single negatively charged residu e (at position L213, across the binding pocket at position L225, or ou tside the pocket at M43) or loss of a Positively charged residue (at p osition M231) is sufficient to restore proton transfer activity to the complex. The proton transport pathways in the suppressor strains cann ot, in principle, be identical to that of the wild type. The apparent mutability of this pathway suggests that the reaction center can serve as a model system to study the structural basis of protein-mediated p roton transport.