Deborah K. Hanson

Initial electron transfer in photosynthetic reaction centers of Rhodobacter capsulatus mutants

Abstract The stimulated emission decay time constants were measured for a series of Rhodobacter capsulatus reaction centers with site-specific mutations at the symmetry related locations M208 and L181. We report the first mutant (Phe L181 → Tyr) that exhibits an initial electron transfer rate faster than the native organism at 295 K, and determine that the tyrosine at position M208 cannot be fully responsible for the unidirectionality of electron transfer.

Electron and proton transfer to the quinones in bacterial photosynthetic reaction centers: Insight from combined approaches of molecular genetics and biophysics

We present here new results together with an overview of the current knowledge on the coupled processes of electron and proton transfer in bacterial reaction centers. The importance of a multidisciplinary approach associating molecular genetics, structural biology, biochemistry and spectroscopy is underlined. We emphasize the electrostatic role of the protein to maintain a negative electrostatic potential near the second quinone electron acceptor in order to: i) accelerate the overall rate of proton transfer from the cytoplasm to this acceptor by increasing the pKs of some groups involved in this process; ii) increase the local proton concentration near this acceptor. We also point out the possibility of long distance propagation of the electrostatic effects through the protein associated with relaxation processes triggered by the formation of the semiquinone anions on the first flash.

Sequences of versatile broad-host-range vectors of the RK2 family.

Plasmid pRK404-a smaller derivative of RK2-is a tetracycline-resistant broad-host-range vector that carries a multiple cloning site and the lacZ(alpha) peptide that enables blue/white selection for cloned inserts in Escherichia coli. We present herein the complete and annotated sequence of pRK404 and three related vectors-pRK437, pRK442, and pRK442(H). These derivatives have proven to be valuable tools for genetic manipulation in Gram-negative bacteria. The knowledge of their complete sequences will facilitate efficient future engineering of them and will enhance their general applicability to the design of genetic systems for use in organisms for which new genomic sequence data are becoming available.

Towards higher-throughput membrane protein production for structural genomics initiatives

Integral membrane proteins present unparalleled challenges for structural genomics programs. Samples from this class of proteins are not only difficult to produce in quantities sufficient for analysis by X-ray diffraction or NMR, but their hydrophobic properties add extra dimension to their purification and subsequent crystallization. New systems that seek to tackle the production problems are in development. In our laboratory, one such strategy exploits the unique physiology of the Rhodobacter species of photosynthetic bacteria where we have designed an overexpression system that coordinates the heterologous production of targeted hydrophobic proteins with nascent, unfilled membranes that can be used to harbor them. In this study, we describe the means by which purification of recombinant membrane proteins produced in such a fashion can be purified efficiently from Rhodobacter membranes using relatively higher-throughput, semi-automated methods. These protocols utilize a state-of-the-art FPLCTM system for affinity chromatography, followed by gel filtration or ion exchange chromatography to enhance purity for crystallization attempts. The Rhodobacter expression system coupled with the semi-automation of purification steps represents an advance towards the development of a strategy for obtaining structures for membrane proteins at a more rapid pace.

Second-site mutation at M43 (Asn→Asp) compensates for the loss of two acidic residues in the QB site of the reaction center.

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.

Evaluation of the photosynthetic reaction center protein for potential use as a bioelectronic circuit element.

The characterization of a bioelectronic composite prepared by molecular wiring of a bacterial photosynthetic reaction center (RC) to a metal (Au) electrode is described. Two unique attachment sites on the protein surface were studied as sites for electrical connections‐a polyhistidine tag introduced by site‐directed mutagenesis and a native cysteine amino acid residue. These two attachment sites were evaluated independently and found to serve effectively in coupling the protein to the electrode surface asymmetrically. Cyclic voltammetry (CV) was used to monitor protein integrity and confirm protein chemisorption and orientation to the organofunctionalized gold electrode. Single‐protein transport measurements made with conductive atomic force microscopy (C‐AFM) were used to study the electrical transport. Current‐voltage (I–V) curves obtained by wiring the protein at the polyhistidine tag showed diodelike behavior. The cysteine attachment site does not serve as an efficient means to address the protein electrically. Scanning tunneling spectroscopy (STS) performed on RCs coupled at the donor side under both dark‐and white‐light‐illuminated conditions confirmed the C‐AFM studies.

Resolution of electron and proton transfer events in the electrochromism associated with quinone reduction in bacterial reaction centers

We have measured the electrochromic response of the bacteriopheophytin, BPh, and bacteriochlorophyll, BChl, cofactors during the QA−QB → QAQB− electron transfer in chromatophores of Rhodobacter (Rb.) capsulatus and Rb. sphaeroides. The electrochromic response rises faster in chromatophores and is more clearly biexponential than it is in isolated reaction centers. The chromatophore spectra can be interpreted in terms of a clear kinetic separation between fast electron transfer and slower non-electron transfer events such as proton transfer or protein relaxation. The electrochromic response to electron transfer exhibits rise times of about 4 µs (70%) and 40 µs (30%) in Rb. capsulatus and 4 µs (60%) and 80 µs (40%) in Rb. sphaeroides. The BPh absorption band is shifted to nearly equivalent positions in the QA− and nascent QB− states, indicating that the electrochromic perturbation of BPh absorption from the newly formed QB− state is comparable to that of QA− . Subsequently, partial attenuation of the QB− electrochromism occurs with a time constant on the order of 200 µs. This can be attributed to partial charge compensation by H+ (or other counter ion) movement into the QB pocket. Electron transfer events were found to be slower in detergent isolated RCs than in chromatophores, more nearly monoexponential, and overlap H+ transfer, suggesting that a change in rate-limiting step has occurred upon detergent solubilization.

Protein Modifications Affecting Triplet Energy Transfer in Bacterial Photosynthetic Reaction Centers

The efficiency of triplet energy transfer from the special pair (P) to the carotenoid (C) in photosynthetic reaction centers (RCs) from a large family of mutant strains has been investigated. The mutants carry substitutions at positions L181 and/or M208 near chlorophyll-based cofactors on the inactive and active sides of the complex, respectively. Light-modulated electron paramagnetic resonance at 10 K, where triplet energy transfer is thermally prohibited, reveals that the mutations do not perturb the electronic distribution of P. At temperatures > or = 70 K, we observe reduced signals from the carotenoid in most of the RCs with L181 substitutions. In particular, triplet transfer efficiency is reduced in all RCs in which a lysine at L181 donates a sixth ligand to the monomeric bacteriochlorophyll B(B). Replacement of the native Tyr at M208 on the active side of the complex with several polar residues increased transfer efficiency. The difference in the efficiencies of transfer in the RCs demonstrates the ability of the protein environment to influence the electronic overlap of the chromophores and thus the thermal barrier for triplet energy transfer.

Protein-chromophore interactions: spectral shifts report the consequences of mutations in the bacterial photosynthetic reaction center

Features of the low temperature absorption spectra of reaction centers from a large family of Rhodobacter capsulatus strains carrying site-specific mutations at the M208Tyr and L181Phe positions are presented. Through systematic analysis of the observed electronic transitions (with accompanying vibronic bands), the primary effects of many of the mutations have been identified and explained in terms of the reaction center structure. Some of the observed shifts are comparable to those that have been seen in other systems as a result of formation of hydrogen bonds or hexacoordination of the central Mg atom in bacteriochlorophyll species. Shifts of the Qy and Qx bacteriochlorophyll bands are also seen as a result of distant mutations near the quinones that change the charge distribution of the reaction center. We include kinetic data which show that the spectral and kinetic characteristics of different cofactors can be modulated depending on the particular amino acid that is substituted at the M208 site.

Temperature and cryoprotectant influence secondary quinone binding position in bacterial reaction centers

We have determined the first de novo position of the secondary quinone QB in the Rhodobacter sphaeroides reaction center (RC) using phases derived by the single wavelength anomalous dispersion method from crystals with selenomethionine substitution. We found that in frozen RC crystals, QB occupies primarily the proximal binding site. In contrast, our room temperature structure showed that QB is largely in the distal position. Both data sets were collected in dark‐adapted conditions. We estimate that the occupancy of the QB site is 80% with a proximal: distal ratio of 4:1 in frozen RC crystals. We could not separate the effect of freezing from the effect of the cryoprotectants ethylene glycol or glycerol. These results could have far‐reaching implications in structure/function studies of electron transfer in the acceptor quinone complex because the above are the most commonly used cryoprotectants in spectroscopic experiments.