The PufX quinone channel enables the light-harvesting 1 antenna to bind more carotenoids for light collection and photoprotection

Photosynthesis in some phototrophic bacteria requires the PufX component of the reaction centre-light-harvesting 1-PufX (RC-LH1-PufX) complex, which creates a pore for quinone/quinol (Q/QH2 ) exchange across the LH1 barrier surrounding the RC. However, photosynthetic bacteria such as Thermochromatium (T.) tepidum do not require PufX because there are fewer carotenoid binding sites, creating multiple pores in the LH1 ring for Q/QH2 exchange. We show that an αTrp-24 →Phe alteration of the Rhodobacter (Rba.) sphaeroides LH1 antenna impairs carotenoid binding and allows photosynthetic growth in the absence of PufX. We propose that acquisition of PufX and confining Q/QH2 traffic to a pore adjacent to the RC QB site is an evolutionary upgrade that allows increased LH1 carotenoid content for enhanced light absorption and photoprotection. This article is protected by copyright. All rights reserved

Absence of the cbb3 terminal oxidase reveals an active oxygen-dependent cyclase involved in bacteriochlorophyll biosynthesis in Rhodobacter sphaeroides.

The characteristic green color associated with chlorophyll pigments results from the formation of an isocyclic fifth ring on the tetrapyrrole macrocyle during the biosynthesis of these important molecules. This reaction is catalyzed by two unrelated cyclase enzymes employing different chemistries. Oxygenic phototrophs such as plants and cyanobacteria utilize an oxygen-dependent enzyme, the major component of which is a diiron protein named AcsF, while BchE, an oxygen-sensitive [4Fe-4S] cluster protein, dominates in phototrophs inhabiting anoxic environments, such as the purple phototrophic bacterium Rhodobacter sphaeroides We identify a potential acsF in this organism and assay for activity of the encoded protein in a strain lacking bchE under various aeration regimes. Initially, cells lacking bchE did not demonstrate AcsF activity under any condition tested. However, on removal of a gene encoding a subunit of the cbb3-type respiratory terminal oxidase, cells cultured under regimes ranging from oxic to microoxic exhibited cyclase activity, confirming the activity of the oxygen-dependent enzyme in this model organism. Potential reasons for the utilization of an oxygen-dependent enzyme in anoxygenic phototrophs are discussed. IMPORTANCE: The formation of the E ring of (bacterio)chlorophyll pigments is the least well-characterized step in their biosynthesis, remaining enigmatic for over 60 years. Two unrelated enzymes catalyze this cyclization step; O2-dependent and O2-independent forms dominate in oxygenic and anoxygenic phototrophs, respectively. We uncover the activity of an O2-dependent enzyme in the anoxygenic purple phototrophic bacterium Rhodobacter sphaeroides, initially by inactivation of the high affinity terminal respiratory oxidase, cytochrome cbb3 We propose that the O2-dependent form allows for the biosynthesis of a low level of bacteriochlorophyll under oxic conditions, so that a rapid initiation of photosynthetic processes is possible for this bacterium upon a reduction of oxygen tension

The C-terminus of PufX plays a key role in dimerisation and assembly of the reaction center light-harvesting 1 complex from Rhodobacter sphaeroides.

In bacterial photosynthesis reaction center-light-harvesting 1 (RC-LH1) complexes trap absorbed solar energy by generating a charge separated state. Subsequent electron and proton transfers form a quinol, destined to diffuse to the cytochrome bc1 complex. In bacteria such as Rhodobacter (Rba.) sphaeroides and Rba. capsulatus the PufX polypeptide creates a channel for quinone/quinol traffic across the LH1 complex that surrounds the RC, and it is therefore essential for photosynthetic growth. PufX also plays a key role in dimerization of the RC-LH1-PufX core complex, and the structure of the Rba. sphaeroides complex shows that the PufX C-terminus, particularly the region from ×49-×53, likely mediates association of core monomers. To investigate this putative interaction we analysed mutations PufX R49L, PufX R53L, PufX R49/53L and PufX G52L by measuring photosynthetic growth, fractionation of detergent-solubilised membranes, formation of 2-D crystals and electron microscopy. We show that these mutations do not affect assembly of PufX within the core or photosynthetic growth but they do prevent dimerization, consistent with predictions from the RC-LH1-PufX structure. We obtained low resolution structures of monomeric core complexes with and without PufX, using electron microscopy of negatively stained single particles and 3D reconstruction; the monomeric complex with PufX corresponds to one half of the dimer structure whereas LH1 completely encloses the RC if the gene encoding PufX is deleted. On the basis of the insights gained from these mutagenesis and structural analyses we propose a sequence for assembly of the dimeric RC-LH1-PufX complex

Mapping the ultrafast flow of harvested solar energy in living photosynthetic cells

Photosynthesis transfers energy efficiently through a series of antenna complexes to the reaction center where charge separation occurs. Energy transfer in vivo is primarily monitored by measuring fluorescence signals from the small fraction of excitations that fail to result in charge separation. Here, we use two-dimensional electronic spectroscopy to follow the entire energy transfer process in a thriving culture of the purple bacteria, Rhodobacter sphaeroides. By removing contributions from scattered light, we extract the dynamics of energy transfer through the dense network of antenna complexes and into the reaction center. Simulations demonstrate that these dynamics constrain the membrane organization into small pools of core antenna complexes that rapidly trap energy absorbed by surrounding peripheral antenna complexes. The rapid trapping and limited back transfer of these excitations lead to transfer efficiencies of 83% and a small functional light-harvesting unit

Quenching Capabilities of Long-Chain Carotenoids in Light-Harvesting-2 Complexes from Rhodobacter sphaeroides with an Engineered Carotenoid Synthesis Pathway

Six light-harvesting-2 complexes (LH2) from genetically modified strains of the purple photosynthetic bacterium Rhodobacter (Rb.) sphaeroides were studied using static and ultrafast optical methods and resonance Raman spectroscopy. These strains were engineered to incorporate carotenoids for which the number of conjugated groups (N = NCC + NCO) varies from 9 to 15. The Rb. sphaeroides strains incorporate their native carotenoids spheroidene (N = 10) and spheroidenone (N = 11), as well as longer-chain analogues including spirilloxanthin (N = 13) and diketospirilloxantion (N = 15) normally found in Rhodospirillum rubrum. Measurements of the properties of the carotenoid first singlet excited state (S1) in antennas from the Rb. sphaeroides set show that carotenoid-bacteriochlorophyll a (BChl a) interactions are similar to those in LH2 complexes from various other bacterial species and thus are not significantly impacted by differences in polypeptide composition. Instead, variations in carotenoid-to-BChl a energy transfer are primarily regulated by the N-determined energy of the carotenoid S1 excited state, which for long-chain (N ≥ 13) carotenoids is not involved in energy transfer. Furthermore, the role of the long-chain carotenoids switches from a lightharvesting supporter (via energy transfer to BChl a) to a quencher of the BChl a S1 excited state B850*. This quenching is manifested as a substantial (∼2-fold) reduction of the B850* lifetime and the B850* fluorescence quantum yield for LH2 housing the longest carotenoids

Characterization Of Human Skin Through Photoacoustic Spectroscopy

The photoacoustic technique is based on the absorption of modulated light by a sample and subsequent heat generation. This generates thermal waves that propagate in the surrounding media According to the Rosencwaig-Gersho Model, such waves produce the pressure oscillation detected as the photoacoustic signal. This technique allows the spectroscopic characterization of multilayer systems: as the thermal diffusion length varies with the modulation frequency of the absorbed light, the depth profile of a sample can be studied by the analysis of the photoacoustic signal at different modulation frequencies. In this work, photoacoustic spectroscopy was used to characterize different human skin samples. Measurements were performed at 70Hz and 17Hz, using a 1000W Xe arc lamp as the light source, for wavelengths between 240nm and 700nm. Skin samples were about 0,5cm diameter. It was possible to obtain the photoacoustic absorption spectra of the stratum corneum and of a deeper layer of epidermis; when the lower modulation frequency is utilized, photoacoustic spectroscopy characterizes the absorption of the whole epidermis, because in this case the thermal diffusion length is thicker than that of the stratum corneum. Photoacoustic spectroscopy was also employed to monitor the drying kinetics of the skin. This was done by analyzing the time evolution of the photoacoustic spectra of skin samples. Pre-treatment of the samples included different periods in a drying chamber. Measurements show that the photoacoustic spectra changes according to the humidity of the skin. Future work includes detailed monitoring of skin hydration.5325136142Morganti, P., Ruocco, E., Wolf, R., Ruocco, V., Percutaneous absorption and delivery systems (2001) Clinics in Dermatology, 19, pp. 489-501Junqueira, L.C., Carneiro, J., (1995) Histologia Básica, 8a Ed., pp. 301-303Bernengo, J.C., Gasquez, C., Falson-Rieg, F., Photoacoustics as a tool for cutaneous permeation studies (1998) High Temperatures-High Pressures, 30, pp. 619-624Gutiérrez-Juárez, G., Vargas-Luna, M., Córdova, T., Varela, J.B., Bernal-Alvarado, J.J., Sosa, M., In vivo measurement of the human skin absorption of toppically applied substances by photoacoustic technique (2002) Physiological Measurement, 23, pp. 1-12Puccetti, G., Lahjomri, F., Leblanc, R.M., Pulsed photoacoustic spectroscopy applied to the diffusion of sunscreen chromophores in human skin: The weakly absorbent regime (1997) Journal of Photochemistry and Photobiology B: Biology, 39, pp. 110-120Vinha, C.A., Haas, U., Qualitative and semiquantitative analysis of dried fruits and seasoning products of paprika using photoacoustic spectroscopy (1997) Journal of Agricultural and Food Chemistry, 45 (4), p. 127

Sunscreen Effects In Skin Analyzed By Photoacoustic Spectroscopy

In the photoacoustic technique, the signal is proportional to the heat produced in a sample as a consequence of modulated light absorption. This technique allows the spectroscopic characterization of multilayer systems: as the thermal diffusion length varies with the light modulation frequency, one can obtain the depth profile of the sample by analyzing the frequency-dependence of the signal. As the photoacoustic signal depends on thermal and optical properties of the sample, structural changes in the system under analysis account for signal variations in time. In this work, photoacoustic spectroscopy was used to characterize samples of sunscreen and the system formed by sunscreen plus skin. Measurements used a 1000W Xe arc lamp as light source, for wavelengths between 240nm and 400nm. This range corresponds to most of the UV radiation that reaches Earth. Skin samples were disks of about 0,5cm diameter. The absorption spectrum of sunscreen was obtained. Finally, photoacoustics was employed to monitor the absorption kinetics of the sunscreen applied to skin samples. This was done by applying sunscreen in a skin sample and recording the photoacoustic spectra in regular time intervals, up to 90 minutes after application. According to measurements, light absorption by the sunscreen plus skin system stabilizes between 25 and 45 minutes after sunscreen application. Results show that this technique can be utilized to monitor drug delivery and farmacokinetics in skin samples.5325143149Barja, P.R., (1996) Estudo da Indução Fotossintética Através da Técnica Fotoacústica: Efeitos de Saturação e Fotoinibição, , (master thesis), IFGW, UNICAMPRosencwaig, A., (1980) Photoacoustics and Photoacoustic Spectroscopy, pp. 219-231. , New York, John Wiley & SonsPucceti, G., Leblanc, R.M., A comparative study on chromophore diffusion inside porous filters by pulsed photoacoustic spectroscopy (1996) Journal of Membrane Science, 119, pp. 213-228Gutiérrez-Juárez, G., Vargas-Luna, M., Córdova, T., In vivo measurement of the human skin absorption of topically applied substances by photoacoustic technique (2002) Physiological Measurement, 23, pp. 1-12Bernengo, J.C., Photoacoustics as a tool for cutaneous permeation studies (1998) High Temperatures-high Pressures, 30, pp. 619-624Azevedo, J.S., UVA/UVB sunscreen determination by second-order derivative ultraviolet spectrophotometry (1999) II Farmaco, 54, pp. 573-578Rettberg, P., Horneck, G., Biologically weighted measurement of UV radiation in space on Earth with the biofilm technique (2000) Advanced Space Research, 26, pp. 2005-2014Van Der Leun, J.C., UV radiation from sunlight: Summary, conclusions and recommendations (1996) Journal of Photochemistry and Photobiology B: Biology, 35, pp. 237-2441Wolf, R., Sunscreens (2001) Clinics in Dermatology, 19, pp. 452-45

Supramolecular bulky phosphines comprising 1,3,5-triaza-7-phosphaadamantane and Zn(salphen)s: structural features and application in hydrosilylation catalysis

International audienceThe use of the commercially available, bifunctional phosphine 1,3,5-triaza-7-phosphaadamantane (abbreviated as PN 3) in conjunction with a series of Zn(salphen) complexes leads to sterically encumbered phosphine ligands as a result of (reversible) coordinative Zn–N interactions. The solid state and solution phase behaviour of these supramolecular ligand systems have been investigated in detail and revealed their stoichiometries in the solid state observed by X-ray crystallography, and those determined in solution by NMR and UV-Vis spectroscopy. Also, upon application of these supramolecular bulky phosphines in hydrosilylation catalysis employing 1-hexene as a substrate, the catalysis data infer the presence of an active Rh species with two coordinated, bulky PN 3 /Zn(salphen) assembly units having a maximum of three Zn(salphen)s associated per PN 3 scaffold, with an excess of bulky phosphines hardly affecting the overall activity