Integral membrane proteins often present daunting challenges for biophysical characterization, a fundamental issue being how to select a surfactant that will optimally preserve the individual structure and functional properties of a given membrane protein. Bacterial reaction centers offer a rare opportunity to compare the properties of an integral membrane protein in different artificial lipid/surfactant environments with those in the native bilayer. Here, we demonstrate that reaction centers purified using a styrene maleic acid copolymer remain associated with a complement of native lipids and do not display the modified functional properties that typically result from detergent solubilization. Direct comparisons show that reaction centers are more stable in this copolymer/lipid environment than in a detergent micelle or even in the native membrane, suggesting a promising new route to exploitation of such photovoltaic integral membrane proteins in device applications.

General rightsThis document is made available in accordance with publisher policies. Please cite only the published version using the reference above. Full terms of use are available: http://www.bristol.ac.uk/pure/about/ebr-terms AbstractIn a quest to fabricate novel solar energy materials, the high quantum efficiency and long charge separated states of photosynthetic pigment-proteins are being exploited through their direct incorporation in bioelectronic devices. In this work, photocurrent generation by bacterial reaction center-light harvesting 1 (RC-LH1) complexes self-assembled on a nanostructured silver substrate yielded a peak photocurrent of 166 µA cm -2 under 1 sun illumination, and a maximum of over 400 µA cm -2 under 4 suns, the highest reported to date.A 2.5-fold plasmonic enhancement of light absorption per RC-LH1 complex was measured on the rough silver substrate. This plasmonic interaction was assessed using confocal fluorescence microscopy, revealing an increase of fluorescence yield and radiative rate of the RC-LH1 complexes. Nano-structuring of the silver substrate also enhanced the stability of the protein under continuous illumination by almost an order of magnitude relative to a nonstructured bulk silver control. Due to its ease of construction, increased protein loading capacity, stability and more efficient use of light, this hybrid material is an excellent candidate for further development of plasmon enhanced biosensors and bio-photovoltaic devices.3

Highlights d A cell organelle, the photosynthetic chromatophore, is modeled in atomistic detail d Segregation of protein complexes tunes chromatophore structure and function d The electrostatic environment of the organelle supports low light-adaptation d Distinct modes of quinone diffusion underpin efficient electron transfer dynamics

The Rhodobacter sphaeroides reaction centre is a relatively robust and tractable membrane protein that has potential for exploitation in technological applications, including biohybrid devices for photovoltaics and biosensing. This report assessed the usefulness of the photocurrent generated by this reaction centre adhered to a small working electrode as the basis for a biosensor for classes of herbicides used extensively for the control of weeds in major agricultural crops. Photocurrent generation was inhibited in a concentration-dependent manner by the triazides atrazine and terbutryn, but not by nitrile or phenylurea herbicides. Measurements of the effects of these herbicides on the kinetics of charge recombination in photo-oxidised reaction centres in solution showed the same selectivity of response. Titrations of reaction centre photocurrents yielded half maximal inhibitory concentrations of 208 nM and 2.1 µM for terbutryn and atrazine, respectively, with limits of detection estimated at around 8 nM and 50 nM, respectively. Photocurrent attenuation provided a direct measure of herbicide concentration, with no need for model-dependent kinetic analysis of the signal used for detection or the use of prohibitively complex instrumentation, and prospects for the use of protein engineering to develop the sensitivity and selectivity of herbicide binding by the Rba. sphaeroides reaction centre are discussed.

The cytochrome b 6 f (cytb 6 f) complex plays a central role in oxygenic photosynthesis, linking electron transfer between photosystems I and II and conserving solar energy as a transmembrane proton gradient for ATP synthesis 1-3. Electron transfer within cytb 6 f occurs via the Q-cycle, which catalyses the oxidation of plastoquinol (PQH 2) and the reduction of both plastocyanin (PC) and plastoquinone (PQ) at two separate sites via electron bifurcation 2. In higher-plants cytb 6 f also acts as a redox-sensing hub, pivotal to the regulation of light harvesting a n d c y c l i c e l e c t r o n t r a n s f e r t h at protect against metabolic and environmental stresses 3. Here we present a 3.6 Å resolution c r y o-e l e c t r o n m i c r o s c o p y (c r y o-E M) structure of the dimeric cytb 6 f complex from spinach, which reveals the structural basis for operation of the Q-cycle and its redox sensing function. The complex contains up to three natively bound PQ molecules. The first, PQ1, is bound to one cytb 6 f monomer at the PQ oxidation site (Q p) adjacent to haem b p and chlorophyll a. Two conformations of the chlorophyll a phytyl tail were resolved, one that prevents access to the Q p site and another that permits it, supporting a gating function for the chlorophyll a involved in redox sensing. PQ2 straddles the intermonomer cavity, partially obstructing the PQ reduction site (Q n) on the PQ1 side and committing the electron transfer network to turnover at the occupied Q n site in the neighbouring monomer. A conformational switch involving the haem c n propionate promotes two-electron, two-proton reduction at the Q n site and avoids formation of the reactive intermediate semiquinone. The location of a tentatively assigned third PQ molecule is consistent with a transition between the Q p and Q n sites in opposite monomers during the Q-cycle. The spinach cytb 6 f structure therefore provides new insights into how the complex fulfils its catalytic and regulatory roles in photosynthesis. Photosynthesis sustains life on Earth by converting light into chemical energy in the form of ATP and NADPH, producing oxygen as a by-product. Two light-powered electron transfer reactions at photosystems I and II (PSI and PSII) are linked via the cytb 6 f complex to form the so-called 'Z-scheme' of photosynthetic linear electron transfer (LET) 1. Cytb 6 f catalyses the rate-limiting step in the LET chain, coupling the oxidation of PQH 2 and reduction of PC and PQ to the generation of a transmembrane proton gradient (p), used by ATP synthase to make ATP 2,3. The cytb 6 f complex is analogous to the cytochrome bc 1 (cytbc 1) complex found in mitochondria 4 and anoxygenic photosynthetic bacteria 5 and both operate via the modified Q

The high quantum efficiency of photosynthetic reaction centers (RCs) makes them attractive for bioelectronic and biophotovoltaic applications. However, much of the native RC efficiency is lost in communication between surface-bound RCs and electrode materials. The state-of-the-art biophotoelectrodes utilizing cytochrome c (cyt c) as a biological wiring agent have at best approached 32% retained RC quantum efficiency. However, bottlenecks in cyt c-mediated electron transfer have not yet been fully elucidated. In this work, protein film voltammetry in conjunction with photoelectrochemistry is used to show that cyt c acts as an electron-funneling antennae that shuttle electrons from a functionalized rough silver electrode to surface-immobilized RCs. The arrangement of the two proteins on the electrode surface is characterized, revealing that RCs attached directly to the electrode via hydrophobic interactions and that a film of six cyt c per RC electrostatically bound to the electrode. We show that the additional electrical connectivity within a film of cyt c improves the high turnover demands of surface-bound RCs. This results in larger photocurrent onset potentials, positively shifted half-wave reduction potentials, and higher photocurrent densities reaching 100 μA cm–2. These findings are fundamental for the optimization of bioelectronics that utilize the ubiquitous cyt c redox proteins as biological wires to exploit electrode-bound enzymes.

Solubilisation of biological lipid bilayer membranes for analysis of their protein complement has traditionally been carried out using detergents, but there is increasing interest in the use of amphiphilic copolymers such as styrene maleic acid (SMA) for the solubilisation, purification and characterisation of integral membrane proteins in the form of protein/lipid nanodiscs. Here we survey the effectiveness of various commercially-available formulations of the SMA copolymer in solubilising Rhodobacter sphaeroides reaction centres (RCs) from photosynthetic membranes. We find that formulations of SMA with a 2:1 or 3:1 ratio of styrene to maleic acid are almost as effective as detergent in solubilising RCs, with the best solubilisation by short chain variants (< 30 kDa weight average molecular weight). The effectiveness of 10 kDa 2:1 and 3:1 formulations of SMA to solubilise RCs gradually declined when genetically-encoded coiled-coil bundles were used to artificially tether normally monomeric RCs into dimeric, trimeric and tetrameric multimers. The ability of SMA to solubilise reaction centre-light harvesting 1 (RC-LH1) complexes from densely packed and highly ordered photosynthetic membranes was uniformly low, but could be increased through a variety of treatments to increase the lipid:protein ratio. However, proteins isolated from such membranes comprised clusters of complexes in small membrane patches rather than individual proteins. We conclude that short-chain 2:1 and 3:1 formulations of SMA are the most effective in solubilising integral membrane proteins, but that solubilisation efficiencies are strongly influenced by the size of the target protein and the density of packing of proteins in the membrane.

The reaction-center light-harvesting complex 1 (RC-LH1) is the core photosynthetic component in purple phototrophic bacteria. We present two cryo–electron microscopy structures of RC-LH1 complexes from Rhodopseudomonas palustris. A 2.65-Å resolution structure of the RC-LH114-W complex consists of an open 14-subunit LH1 ring surrounding the RC interrupted by protein-W, whereas the complex without protein-W at 2.80-Å resolution comprises an RC completely encircled by a closed, 16-subunit LH1 ring. Comparison of these structures provides insights into quinone dynamics within RC-LH1 complexes, including a previously unidentified conformational change upon quinone binding at the RC QB site, and the locations of accessory quinone binding sites that aid their delivery to the RC. The structurally unique protein-W prevents LH1 ring closure, creating a channel for accelerated quinone/quinol exchange.