No abstract

No abstract

In photosynthetic reaction centers, a quinone molecule, QB, is the terminal acceptor in light-induced electron ransfer. The protonatable residues Glu-L212 and have been implicated in the binding of QB and in proton tr_aner 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 liht-induced electron or proton transfer in Rhodobacter capsuadaus. 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 bindin 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.The reaction center (RC) in the photosynthetic membrane mediates light-initiated redox chemistry, producing a transmembrane charge separation (for review, see ref. 1). The RC from purple bacteria is the first integral membrane protein complex for which an atomic structure has been determined (1-4). Thus, the RC currently serves as the best model for understanding protein-mediated electron transfer and the subsequent transfer of protons from the aqueous phase to buried protein sites.The RC is a complex of three protein subunits, the intermembrane L and M chains and the H polypeptide, as well as several cofactors (four bacteriochlorophylls, two bacteriopheophytins, two quinones, and a nonheme iron atom). The cofactors are chemically active in light-induced electron transfer (for review, see ref. 5) and the detailed contribution of the protein to the electrochemistry of this process is obscure. An approximate two-fold symmetry relates the L and M subunits and their cofactors, suggesting two possible routes of charge separation across the photosynthetic membrane. However, normal photochemistry follows only the pathway that is primarily associated with the L subunit.The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.Upon light activation, an electron is transferred from the "special ...

Two acidic residues, L212Glu and L213Asp, in the QB binding sites of the photosynthetic reaction centers of Rhodobacter capsulatus and Rhodobacter sphaeroides are thought to play central roles in the transfer of protons to the quinone anion(s) generated by photoinduced electron transfer. We constructed the site-specific double mutant L212Ala-L213Ala in R. capsulatus, that is incapable of growth under photosynthetic conditions. A photocompetent derivative of that strain has been isolated that carries the original L212Ala-L213Ala double mutation and a second-site suppressor mutation at residue M43 (Asn→Asp), outside of the QB binding site, that is solely responsible for restoring the photosynthetic phenotype. The Asp,Asn combination of residues at the L213 and M43 positions is conserved in the five species of photosynthetic bacteria whose reaction center sequences are known. In R. capsulatus and R. sphaeroides, the pair is L213Asp-M43Asn. But, the reaction centers of Rhodopseudomonas viridis, Rhodospirillum rubrum and Chloroflexus aurantiacus reverse the combination to L213Asn-M43Asp. In this respect, the QB site of the suppressor strain resembles that of the latter three species in that it couples an uncharged residue at L213 with an acidic residue at M43. These reaction centers, in which L213 is an amide, must employ an alternative proton transfer pathway. The observation that the M43Asn→Asp mutation in R. capsulatus compensates for the loss of both acidic residues at L212 and L213 suggests that M43Asp is involved in a new proton transfer route in this species that resembles the one normally used in reaction centers of Rps. virddis, Rsp. rubrum and C. aurantiacus.

Using the ISO-DALT system for two-dimensional (2-D) electrophoresis and the TYCHO system for computer analysis of the resulting protein maps, we obtained high quality quantitative protein abundance data from Coomassie Brilliant Bluestained gelsofmouseliver samples. High resolution gels allow more than I00 proteins to be measured with coefficients of variation less than 15 %. A comparison of results from two mouse strains (C57BL/6 and BALB/c) and the cross between them (BCF ,) shows that a large number of qutive polymorphisms can be detected, and that, as expected, the amount of protein produced in the heterozygote is intermediate between the parental values. The system described is shown to be capable of reliably detecting decreases in protein abundance such as those expected to result from radiation-induced deletion of one copy of a gene. The implications of these results for the study of gene regulation are discussed in relation to applications in genetics, toxicology, and differentiation.