title: Site-specific and compensatory mutations imply unexpected pathways for proton delivery to the QB binding site of the photosynthetic reaction center.
abstract: In photosynthetic reaction centers, a quinone molecule, QB, is the terminal acceptor in light-induced electron transfer. The protonatable residues Glu-L212 and Asp-L213 have been implicated in the binding of QB and in proton transfer to QB anions generated by electron transfer from the primary quinone QA. Here we report the details of the construction of the Ala-L212/Ala-L213 double mutant strain by site-specific mutagenesis and show that its photosynthetic incompetence is due to an inability to deliver protons to the QB anions. We also report the isolation and biophysical characterization of a collection of revertant and suppressor strains that have regained the photosynthetic phenotype. The compensatory mutations that restore function are diverse and show that neither Glu-L212 nor Asp-L213 is essential for efficient light-induced electron or proton transfer in Rhodobacter capsulatus. Second-site mutations, located within the QB binding pocket or at more distant sites, can compensate for mutations at L212 and L213 to restore photocompetence. Acquisition of a single negatively charged residue (at position L213, across the binding pocket at position L225, or outside the pocket at M43) or loss of a positively charged residue (at position M231) is sufficient to restore proton transfer activity to the complex. The proton transport pathways in the suppressor strains cannot, 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 proton transport.
authors.affiliation: Biological and Medical Research Division, Argonne National Laboratory, IL 60439.
authors.forename: D K
authors.initials: DK
authors.lastname: Hanson
authors.forename: D M
authors.initials: DM
authors.lastname: Tiede
authors.forename: S L
authors.initials: SL
authors.lastname: Nance
authors.forename: C H
authors.initials: CH
authors.lastname: Chang
authors.forename: M
authors.initials: M
authors.lastname: Schiffer
mesh_terms: D000409:Alanine; D000595:Amino Acid Sequence; D001224:Aspartic Acid; D016227:Benzoquinones / Q000378:metabolism*; D001665:Binding Sites; D004579:Electron Transport; D005971:Glutamates; D018698:Glutamic Acid; D007700:Kinetics; D016297:Mutagenesis, Site-Directed; D045322:Photosynthetic Reaction Center Complex Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D011487:Protein Conformation; D016354:Rhodobacter capsulatus / Q000378:metabolism*; D013053:Spectrophotometry
chemical_list: D016227:Benzoquinones; D005971:Glutamates; D045322:Photosynthetic Reaction Center Complex Proteins; D001224:Aspartic Acid; D018698:Glutamic Acid; C004532:quinone; D000409:Alanine