A Genetic System for Rapidly Assessing Herbicides That Compete for the Quinone Binding Site of Photosynthetic Reaction Centers

Bylina, Edward J.; Jovine, Raffael V. M.; Youvan, Douglas C.

doi:10.1038/nbt0189-69

Cited by 56 publications

(35 citation statements)

References 23 publications

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“…The mutations were verified by dideoxynucleotide sequencing of double-stranded DNA (Sequenase, United States Biochemical). The mutant L gene was returned to the pufoperon in plasmids pU29 (22) and pU2922 (23). Mutant plasmids were transferred to Rb.…”

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confidence: 99%

“…The system ofplasmids described by Bylina et al (22,23) was used for site-specific mutagenesis according to a kit (Bio-Rad) based on the method of Kunkel et al (24). The HindIII-Kpn I fragment carrying residues 1-253 of the L gene was subcloned into bifunctional vector pBS+/-(Stratagene) and mutagenized with a 30-mer containing two single-base changes at L212 and L213.…”

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Site-specific and compensatory mutations imply unexpected pathways for proton delivery to the QB binding site of the photosynthetic reaction center.

Hanson

Tiede

Nance

et al. 1993

Proc. Natl. Acad. Sci. U.S.A.

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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 ...

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Site-specific and compensatory mutations imply unexpected pathways for proton delivery to the QB binding site of the photosynthetic reaction center.

Hanson

Tiede

Nance

et al. 1993

Proc. Natl. Acad. Sci. U.S.A.

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“…As shown in Fig. 4, all mutations confering herbicide resistances described until now in purple bacteria are located in helix DE, between helices DE and E and in helix E [6,9,18,19]. The mutation we have found in this ter r mutant is the first one located in this region of the QB pocket.…”

Section: Discussionmentioning

confidence: 56%

A new mutation in the pufL gene responsible for the terbutryn resistance phenotype in Rubrivivax gelatinosus

1995

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“…A system to construct and express mutants of the L and M genes of the polycistronic, oxygen-regulated puf operon of R. capsulatus was developed previously (47)(48)(49). The expression host strain, U43, does not synthesize any light-harvesting antennae or RC complexes of the photosynthetic apparatus (i.e.…”

Section: Resultsmentioning

confidence: 99%

High Throughput Engineering to Revitalize a Vestigial Electron Transfer Pathway in Bacterial Photosynthetic Reaction Centers

Faries

Kressel

Wander

et al. 2012

Journal of Biological Chemistry

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Background: Bacterial reaction centers catalyze light-induced transmembrane electron transport using only one of two chemically equivalent pathways. Results: Multiplexed screening uncovers semirandom mutations that unexpectedly activate the unused pathway by employing ionizable residues. Conclusion: High throughput mutagenesis approaches reveal structure/function relationships that govern electron transfer efficiency. Significance: Directed molecular evolution can reveal principles that enable efficient, unidirectional, transmembrane electron transfer for the design of de novo pathways or biomimetic devices.

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A Genetic System for Rapidly Assessing Herbicides That Compete for the Quinone Binding Site of Photosynthetic Reaction Centers

Cited by 56 publications

References 23 publications

Site-specific and compensatory mutations imply unexpected pathways for proton delivery to the QB binding site of the photosynthetic reaction center.

Site-specific and compensatory mutations imply unexpected pathways for proton delivery to the QB binding site of the photosynthetic reaction center.

A new mutation in the pufL gene responsible for the terbutryn resistance phenotype in Rubrivivax gelatinosus

High Throughput Engineering to Revitalize a Vestigial Electron Transfer Pathway in Bacterial Photosynthetic Reaction Centers

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