Photosynthetic unit size and electron-transport chain in a photoreaction center-depleted mutant of Rhodospirillum rubrum

The aim of this work was to explain the relatively fast growth of a mutant of Rhodospirillum rubrum (F24.1) which contains 7–8% of an apparently normal photoreaction center. We explored the double hypothesis that the size of its photosynthetic unit is larger than that of the wild type and that its electron-transport chain is organized in a network rather than in isolated loops. The first feature would allow faster growth under less than saturating light intensities and the second would allow faster maximal electron fluxes than would be predicted from the photoreaction center content. With respect to the first possibility, measurements of absorbance changes at 793 nm induced by short flashes of increasing intensity indicate that the photosynthetic unit of strain F24.1 is 5.6-fold larger than that of strain S1. The second possibility was verified by measuring relative electron fluxes at the photoreaction center in the two strains. This was established in the steady state from the amount of primary donor oxidized by a continuous light beam of increasing intensity. This electron flux was found to be about 70% as high in strain F24.1 as in strain S1. A more detailed study of the electron-transport chain indicated that cytochrome c₂ is by far the main secondary electron donor in strain F24.1. No evidence could be obtained for the existence of another secondary donor in that strain. The mole ratio of cytochrome c₂ to photoreaction center is about 6 in strain F24.1 as conpared to about 0.5 in strain S1. In strain 24.1, the pool of secondary donor appears to be collectively involved in the reduction of the oxidized primary donor. The replacement time at the photoreaction center of a first equivalent of oxidized cytochrome c₂ by a second equivalent of reduced cytochrome c₂ is less than or equal to 0.2 ms. The effect of the photoreaction center content on the size of the photosynthetic unit is discussed in terms of the different models proposed for the organisation of the photosynthetic unit. We propose that the electron-transport chain is organized in a network, perhaps by virtue of the lateral mobility of some of the electron carriers such as ubiquinone and cytochrome c₂.     

PCR-RFLP and AP-PCR of rbcL and ITS of rDNA Show That ×Taxodiomeria peizhongii (Taxodium×Cryptomeria) Is not an Intergeneric Hybrid

×Taxodiomeria peizhongii Z. J. Ye, J. J. Zhang et S. H. Pan was regarded as a new intergeneric hybrid between Taxodium mucronatum Tenore (as the female donor) and Cryptomeria fortunei Hooibrenk ex Otto et Dietr (as the male donor). To confirm the authenticity of the intergeneric hybrid, we analyzed the rbcL gene and the internal transcribed spacer (ITS) of 26S‐18S ribosomal RNA gene of the three species using polymerase chain reaction‐restriction fragment length polymorphism (PCR‐RFLP) and arbitrarily primed PCR (AP‐PCR), and obtained the following results: i) Taxodiomeria peizhongii had the same RFLP maps of the rbcL gene and the ITS as Taxodium mucronatum, but was different from C. fortunei; ii) a 311‐bp PCR amplification product was obtained in C. fortunei by AP‐PCR of ITS, but was not found in Taxodiomeria peizhongii. Our results have demonstrated that C. fortunei did not provide any genome for Taxodiomeria peizhongii, implying that T. peizhongii is not an intergeneric hybrid between the two species. (Managing editor: Wei Wang)     

Effect of inhibitors,redox state and isoprenoid chain length on the affinity of ubiquinone for the secondary acceptor binding site in the reaction centers of photosynthetic bacteria

Quinone and inhibitor binding to Rhodopseudomonas sphaeroides (R-26 and GA) reaction centers were studied using spectroscopic methods and by direct adsorption of reaction centers onto anion exchange filters in the presence of ¹⁴C-labelled quinone or inhibitor. These measurements show that as secondary acceptor, Q_B, ubiquinone (UQ) is tightly bound in the semiquinone form and loosely bound in the quinone and quinol forms. The quinol is probably more loosely bound than the quinone. o-Phenanthroline and terbutryn, a triazine inhibitor, compete with UQ and with each other for binding to the reaction center. Inhibition by o-phenanthroline of electron transfer from the primary to the secondary quinone acceptor (Q_A to Q_B) occurs via displacement of UQ from the Q_B binding site. Displacement of UQ by terbutryn is apparently accessory to the inhibition of electron transfer. Terbutryn binding is lowered by reduction of Q_B to Q^?_B but is practically unaffected by reduction of Q_A to Q^?_A in the absence of Q_B. UQ-9 and UQ-10 have a 5- to 6-fold higher binding affinity to the Q_B site than does UQ-1, indicating that the long isoprenoid chain facilitates the binding to the Q_B site.     

Circular dichroism spectra of oriented photoreaction center from Rhodospirillum rubrum

The circular dichroism spectra of oriented and unoriented photoreaction centers of Rhodospirillum rubrum are compared. Orientation is achieved by pressing photoreaction center suspended in polyacrylamide gel. The biphasic bands at 870 and 810 nm and at 630 and 600 nm undergo a rotatory strength decrease when measured in the direction of the pressure, but not when measured in the direction normal to the pressure. Such a decrease in oriented photoreaction center is consistent with the model according to which these bands are dimer exciton bands of the special pair bacteriochlorophyll.     

ARBRE: Computational resource to predict pathways towards industrially important aromatic compounds

Synthetic biology and metabolic engineering rely on computational search tools for predictions of novel biosynthetic pathways to industrially important compounds, many of which are derived from aromatic amino acids. Pathway search tools vary in their scope of covered reactions and compounds, as well as in metrics for ranking and evaluation. In this work, we present a new computational resource called ARBRE: Aromatic compounds RetroBiosynthesis Repository and Explorer. It consists of a comprehensive biochemical reaction network centered around aromatic amino acid biosynthesis and a computational toolbox for navigating this network. ARBRE encompasses over 33′000 known and 390′000 novel reactions predicted with generalized enzymatic reactions rules and over 74′000 compounds, of which 19′000 are known to biochemical databases and 55′000 only to PubChem. Over 1′000 molecules that were solely part of the PubChem database before and were previously impossible to integrate into a biochemical network are included into the ARBRE reaction network by assigning enzymatic reactions. ARBRE can be applied for pathway search, enzyme annotation, pathway ranking, visualization, and network expansion around known biochemical pathways and products of lignin degradation to predict valuable compound derivations. In line with the standards of open science, we have made the toolbox freely available to the scientific community on git (https://github.com/EPFL-LCSB/ARBRE) and we provide the web-version at http://lcsb-databases.epfl.ch/arbre/. We envision that ARBRE will provide the community with a new computational resource and comprehensive search tool to predict and rank pathways towards industrially important aromatic compounds.     

Reaction Rates in Organic Media Show Similar Dependence on Water Activity with Lipase Catalyst Immobilized on Different Supports

Lipase (E.C. 3.1.1.3) from Rhizomucor miehei was adsorbed on silica, zirconia and five alumina support materials. The immobilised preparations were used to catalyse esterincation reactions of decanoic acid and dodecanol in hexane. The immobilised lipase and the organic phase were separately preequilibrated to the desired water activities. The various support materials adsorbed widely different amounts of water at a given water activity. The reaction rates with all the support materials show similar dependence on water activity when the rates were normalised with the optimal rate for that support material. Hence water activity predicts the optimal conditions much better than water content.     

Labeling of chromatophore membranes and reaction centers from the photosynthetic bacterium Rhodospirillum rubrum with the hydrophobic marker 5-[125I]iodonaphthyl-1-azide

Chromatophores of the photosynthetic bacterium Rhodospirillum rubrum and isolated reaction centers were labeled with the lipophilic membrane marker 5-[¹²⁵I]iodonaphthyl-1-azide. The two smaller reaction center proteins L and M bind more label than the larger subunit H, a fact supporting the proposed localisation of the 3 subunits obtained with hydrophilic labels. Besides these integral proteins the lipids, among them mainly the pigments and the quinones, are highly labeled suggesting a hydrophobic environment around these molecules and a preferred reactivity to iodonaphthylazide. Such a hydrophobic environment may be of great importance for the function of the photosynthetic reaction centers especially for the charge separation and the primary reactions in electron transport.     

Kinetics and thermodynamics of the P870+Q−A → P870+Q−B reaction in isolated reaction centers from the photosynthetic bacterium Rhodopseudomonas sphaeroides

The temperature dependences of the P870⁺Q^?_A → P870Q_A and P870⁺Q^?_B → P870Q_B recombination reactions were measured in reaction centers from Rhodopseudomonas sphaeroides. The data indicate that the P870⁺Q^?_B state decays by thermal repopulation of the P870⁺Q^?_A state, followed by recombination. ΔG° for the P870⁺Q^?_A → P870⁺Q^?_B reaction is ?6.89 kJ · mol^?1, while ΔH° = ?14.45 kJ · mol^?1 and ?TΔS° = + 7.53 kJ · mol^?1. The activation ethalpy, $\text{H}{}^3$, for the P870⁺Q^?_A Δ P870⁺Q^?_B reaction is +56.9 kJ · mol^?1, while the activation entropy is near zero. The results permit an estimate of the shape of the potential energy curve for the P870⁺Q^?_A → P870⁺Q^?_B electron transfer reaction.     

Analysis by exciton theory of the optical properties of the Chloroflexus aurantiacus reaction center

The optical properties of the reaction center of the filamentous green bacterium Chloroflexus aurantiacus, that contains three bacteriochlorophyll (BChl) a and three bacteriopheophytin (BPh) a molecules, were analyzed in the near-infrared region with the aid of exciton theory. The coordinates obtained from the X-ray analysis of the reaction center of Rhodopseudomonas viridis (Deisenhofer, J., Epp, O., Miki, K., Huber, R. and Michel, H. (1984) J. Mol. Biol. 180, 385–398) were used for the geometry of the reaction center of C. aurantiacus, with the replacement of one of the ‘accessory’ BChl molecules by BPh. The results were found to be in good agreement with experimental low-temperature absorption spectra, linear and circular dichroism and fluorescence polarization spectra and lead to the following conclusions. The allowed, low-energy exciton transition of the primary electron donor (P-865) is located at 887 nm and carries the dipole strength of approx. two BChl a monomers; the high-energy exciton transition, around 790 nm, is mixed with wave functions of other pigments, which explains its relatively small angle with respect to the 887 nm transition. The optical transition of the accessory BChl a molecule near 812 nm has some contribution of the BChls that constitute P-865. This can account for the experimentally observed reorientation and shift of this transition upon oxidation of P-865. Two of the BPh molecules are located on the same (probably the M) polypeptide subunit and show a clear splitting of absorption bands (11 nm) due to exciton coupling; the single BPh on the opposite branch shows hardly any exciton shift. Similar calculations for reaction centers of purple bacteria that contain four BChl a and two BPh a molecules resulted in a very low dipole strength for the high-energy transition of the primary donor due to antisymmetric mixing with both accessory BChl a wave functions and gave very little splitting of the absorption bands of BPh a. Our results indicate that the arrangement of the chromophores in reaction centers of C. aurantiacus is very similar to that in purple bacteria. The functional L-chains of the reaction centers of purple and filamentous green bacteria consist of pigments of the same type in a probably very similar arrangement.