• Energy Research

RC-LH1-PufX complexes from a genetically modified strain of Rhodobacter sphaeroides that accumulates carotenoids with very long conjugation were studied by ultrafast transient absorption spectroscopy. The complexes predominantly bind the carotenoid diketospirilloxanthin, constituting about 75% of the total carotenoids, which has 13 conjugated C=C bonds, and the conjugation is further extended to two terminal keto groups. Excitation of diketospirilloxanthin in the RC-LH1-PufX complex demonstrates fully functional energy transfer from diketospirilloxanthin to BChl a in the LH1 antenna. As for other purple bacterial LH complexes having carotenoids with long conjugation, the main energy transfer route is via the S2-Qx pathway. However, in contrast to LH2 complexes binding diketospirilloxanthin, in RC-LH1-PufX we observe an additional, minor energy transfer pathway associated with the S1 state of diketospirilloxanthin. By comparing the spectral properties of the S1 state of diketospirilloxanthin in solution, in LH2, and in RC-LH1-PufX, we propose that the carotenoid-binding site in RC-LH1-PufX activates the ICT state of diketospirilloxanthin, resulting in the opening of a minor S1/ICT-mediated energy transfer channel.

The X-ray crystal structure of the Rhodopseudomonas (Rps.) palustris reaction center-light harvesting 1 (RC-LH1) core complex revealed the presence of a sixth protein component, variably referred to in the literature as helix W, subunit W or protein W. The position of this protein prevents closure of the LH1 ring, possibly to allow diffusion of ubiquinone/ubiquinol between the RC and the cytochrome bc1 complex in analogous fashion to the well-studied PufX protein from Rhodobacter sphaeroides. The identity and function of helix W have remained unknown for over 13years; here we use a combination of biochemistry, mass spectrometry, molecular genetics and electron microscopy to identify this protein as RPA4402 in Rps. palustris CGA009. Protein W shares key conserved sequence features with PufX homologs, and although a deletion mutant was able to grow under photosynthetic conditions with no discernible phenotype, we show that a tagged version of protein W pulls down the RC-LH1 complex. Protein W is not encoded in the photosynthesis gene cluster and our data indicate that only approximately 10% of wild-type Rps. palustris core complexes contain this non-essential subunit; functional and evolutionary consequences of this observation are discussed. The ability to purify uniform RC-LH1 and RC-LH1-protein W preparations will also be beneficial for future structural studies of these bacterial core complexes.

Six variants of the LH2 antenna complex from Rba. sphaeroides, comprising the native B800-B850, B800-free LH2 (B850) and four LH2s with various (bacterio)chlorophylls reconstituted into the B800 site, have been investigated with static and time-resolved optical spectroscopies at room temperature and at 77 K. The study particularly focused on how reconstitution of a non-native (bacterio)chlorophylls affects excitation energy transfer between the naturally bound carotenoid spheroidene and artificially substituted pigments in the B800 site. Results demonstrate there is no apparent trend in the overall energy transfer rate from spheroidene to B850 bacteriochlorophyll a; however, a trend in energy transfer rate from the spheroidene S1 state to Qy of the B800 (bacterio)chlorophylls is noticeable. These outcomes were applied to test the validity of previously proposed energy values of the spheroidene S1 state, supporting a value in the vicinity of 13,400 cm-1 (746 nm).

Significance Photosynthesis uses carotenoids as light-harvesting pigments and for photoprotective energy dissipation. The carbon–carbon double bond conjugation length of carotenoids ( N ) affects the carotenoid-to-(bacterio)chlorophyll energy transfer efficiency, but the photoprotective capability was considered to be independent of N . Using light-harvesting complex 2 from the model photosynthetic bacterium Rhodobacter sphaeroides containing ζ-carotene ( N = 7) or neurosporene ( N = 9), we demonstrate that decreasing the conjugation length increases the carotenoid-to-bacteriochlorophyll energy transfer efficiency, in the case of ζ-carotene to ∼100%. However, unity quantum efficiency comes at the cost of photoprotection, suggesting that naturally evolved photosynthesis tolerates some energetic loss to allow essential energy dissipation, explaining why longer-conjugation length carotenoids are utilized in native pigment–protein complexes.

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 208nM and 2.1µM for terbutryn and atrazine, respectively, with limits of detection estimated at around 8nM and 50nM, 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.

Small diffusible redox proteins play a ubiquitous role in bioenergetic systems, facilitating electron transfer (ET) between membrane bound complexes. Sustaining high ET turnover rates requires that the association between extrinsic and membrane-bound partners is highly specific, yet also sufficiently weak to promote rapid post-ET separation. In oxygenic photosynthesis the small soluble electron carrier protein plastocyanin (Pc) shuttles electrons between the membrane integral cytochrome b6f (cytb6f) and photosystem I (PSI) complexes. Here we use peak-force quantitative nanomechanical mapping (PF-QNM) atomic force microscopy (AFM) to quantify the dynamic forces involved in transient interactions between cognate ET partners. An AFM probe functionalised with Pc molecules is brought into contact with cytb6f complexes, immobilised on a planar silicon surface. PF-QNM interrogates the unbinding force of the cytb6f-Pc interactions at the single molecule level with picoNewton force resolution and on a time scale comparable to the ET time in vivo (ca. 120 μs). Using this approach, we show that although the unbinding force remains unchanged the interaction frequency increases over five-fold when Pc and cytb6f are in opposite redox states, so complementary charges on the cytb6f and Pc cofactors likely contribute to the electrostatic forces that initiate formation of the ET complex. These results suggest that formation of the docking interface is under redox state control, which lowers the probability of unproductive encounters between Pc and cytb6f molecules in the same redox state, ensuring the efficiency and directionality of this central reaction in the 'Z-scheme' of photosynthetic ET.

Cytochrome bc 1 complexes are ubiquinol:cytochrome c oxidoreductases, and as such, they are centrally important components of respiratory and photosynthetic electron transfer chains in many species of bacteria and in mitochondria. The minimal complex has three catalytic components, which are cytochrome b , cytochrome c 1 , and the Rieske iron–sulfur subunit, but the function of mitochondrial cytochrome bc 1 complexes is modified by up to eight supernumerary subunits. The cytochrome bc 1 complex from the purple phototrophic bacterium Rhodobacter sphaeroides has a single supernumerary subunit called subunit IV, which is absent from current structures of the complex. In this work we use the styrene–maleic acid copolymer to purify the R. sphaeroides cytochrome bc 1 complex in native lipid nanodiscs, which retains the labile subunit IV, annular lipids, and natively bound quinones. The catalytic activity of the four-subunit cytochrome bc 1 complex is threefold higher than that of the complex lacking subunit IV. To understand the role of subunit IV, we determined the structure of the four-subunit complex at 2.9 Å using single particle cryogenic electron microscopy. The structure shows the position of the transmembrane domain of subunit IV, which lies across the transmembrane helices of the Rieske and cytochrome c 1 subunits. We observe a quinone at the Q o quinone-binding site and show that occupancy of this site is linked to conformational changes in the Rieske head domain during catalysis. Twelve lipids were structurally resolved, making contacts with the Rieske and cytochrome b subunits, with some spanning both of the two monomers that make up the dimeric complex.