Laura Baciou

Charge separation induces conformational changes in the photosynthetic reaction centre of purple bacteria

X-ray structures of the wild-type reaction centre from Rhodobacter sphaeroides have been determined to a resolution of 1.87 A in the neutral (dark) state and to 2.06 A in the charge-separated (light-excited) state. Whereas the overall protein structures of both states are rather similar, the domain around the secondary quinone shows significant shifts. The quinone molecule itself is observed at two different positions. In the neutral state, 55% of the quinone is located distally and 45% proximally to the cytoplasmic side. After excitation by light, however, at least 90% of the quinone is found at the proximal position. Results presented by Stowell et al. (1997) are confirmed, but the quality of crystallographic data has been improved. We compare the data with the structure of the mutant RC L 209 PY that keeps the Q(B) molecule in the proximal position even in the charge-neutral state.

Study of wild type and genetically modified reaction centers from Rhodobacter capsulatus: structural comparison with Rhodopseudomonas viridis and Rhodobacter sphaeroides

Reaction centers from the purple bacterium Rhodobacter (Rb.) capsulatus and from two mutants ThrL226-->Ala and IleL229-->Ser, modified in the binding protein pocket of the secondary quinone acceptor (QB), have been studied by flash-induced absorbance spectroscopy. In ThrL226-->Ala, the binding affinities for endogenous QB (ubiquinone 10) and UQ6 are found to be two to three times as high as the wild type. In contrast, in IleL229-->Ser, the binding affinity for UQ6 is decreased about three times compared to the wild type. In ThrL226-->Ala, a markedly increased sensitivity (approximately 30 times) to o-phenanthroline is observed. In Rhodopseudomonas viridis, where Ala is naturally in position L226, the sensitivity to o-phenanthroline is close to that observed in ThrL226-->Ala. We propose that the presence of Ala in position L226 is responsible for the high sensitivity to that inhibitor. The pH dependencies of the rate constants of P+QB- (kBP) charge recombination kinetics (P is a dimer of bacteriochlorophyll, and QB is the secondary quinone electron acceptor) show destabilization of QB- in ThrL226-->Ala and IleL229-->Ser, compared to the wild type. At low pH, similar apparent pK values of protonation of amino acids around QB- are measured in the wild type and the mutants. In contrast to Rb. sphaeroides, in the wild type Rb. capsulatus, kBP substantially increases in the pH range 7-10. This may reflect some differences in the respective structures of both strains or, alternatively, may be due to deprotonation of TyrL 215 in Rb. capsulatus. At pH 7, measurements of the rate constant of QA to QB electron transfer reveal a threefold greater rate in the reaction centers from wild type Rb. capsulatus (65 +/- 1 0 ps)-1 compared to Rb. sphaeroides.We suggest that this may arise from a 0.7-A smaller distance between the quinones in the former strain. Our spectroscopic data on the wild type Rb. capsulatus reaction center suggest the existence of notable differences with the Rb. sphaeroides reaction center structure.

trans Arachidonic acid isomers inhibit NADPH-oxidase activity by direct interaction with enzyme components

NADPH-oxidase is an enzyme that represents, when activated, the major source of non-mitochondrial reactive oxygen species. In phagocytes, this production is an indispensable event for the destruction of engulfed pathogens. The functional NADPH-oxidase complex consists of a catalytic membrane flavocytochrome b (Cytb(558)) and four cytosolic proteins p47(phox), p67(phox), Rac and p40(phox). The NADPH-oxidase activity is finely regulated spatially and temporally by cellular signaling events that trigger the translocation of the cytosolic subunits to its membrane partner involving post-translational modifications and activation by second messengers such as arachidonic acid (AA). Arachidonic acid in its natural cis-poly unsaturated form (C20:4) has been described to be an efficient activator of the enzyme in vivo and in vitro. In this work, we examined in a cell-free system whether a change of the natural cis geometry to the trans configuration, which could occur either by diet or be produced by the action of free radicals, may have consequences on the functioning of NADPH-oxidase. We showed the inability of mono-trans AA isomers to activate the NADPH-oxidase complex and demonstrated the inhibitory effect on the cis-AA-induced NADPH oxidase activation. The inhibition is mediated by a direct effect of the mono-trans AA which targets both the membrane fraction containing the cytb(558) and the cytosolic p67(phox). Our results suggest that the loss of the natural geometric feature (cis-AA) induces substantial structural modifications of p67(phox) that prevent its translocation to the complex.

Key role of proline L209 in connecting the distant quinone pockets in the reaction center of Rhodobacter sphaeroides

Photosynthetic bacterial reaction centers convert light excitation into chemical free energy. The initial electron transfer leads to the consecutive semireductions of the primary (QA) and secondary (QB) quinone acceptors. The Q\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \setlength{\oddsidemargin}{-69pt} \begin{document} \begin{equation*}{\mathrm{_{A}^{-}}}\end{equation*}\end{document} and Q\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \setlength{\oddsidemargin}{-69pt} \begin{document} \begin{equation*}{\mathrm{_{B}^{-}}}\end{equation*}\end{document} formations induce proton uptake from the bulk. Their magnitudes (H+/Q\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \setlength{\oddsidemargin}{-69pt} \begin{document} \begin{equation*}{\mathrm{_{A}^{-}}}\end{equation*}\end{document} and H+/Q\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \setlength{\oddsidemargin}{-69pt} \begin{document} \begin{equation*}{\mathrm{_{B}^{-}}}\end{equation*}\end{document}, respectively) probe the electrostatic interactions within the complex. The pH dependence of H+/Q\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \setlength{\oddsidemargin}{-69pt} \begin{document} \begin{equation*}{\mathrm{_{A}^{-}}}\end{equation*}\end{document} and H+/Q\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \setlength{\oddsidemargin}{-69pt} \begin{document} \begin{equation*}{\mathrm{_{B}^{-}}}\end{equation*}\end{document} were studied in five single mutants modified at the L209 site (L209P→F,Y,W,E,T). This residue is situated at the border of a continuous chain of water molecules connecting QB to the bulk. In the wild type (WT), a proton uptake band is present at high pH in the H+/Q\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \setlength{\oddsidemargin}{-69pt} \begin{document} \begin{equation*}{\mathrm{_{A}^{-}}}\end{equation*}\end{document} and H+/Q\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \setlength{\oddsidemargin}{-69pt} \begin{document} \begin{equation*}{\mathrm{_{B}^{-}}}\end{equation*}\end{document} curves and is commonly attributed to a cluster of acidic groups situated nearby QB. In the H+/Q\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \setlength{\oddsidemargin}{-69pt} \begin{document} \begin{equation*}{\mathrm{_{A}^{-}}}\end{equation*}\end{document} curves of the L209 variants, this band is systematically absent but remains in the H+/Q\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \setlength{\oddsidemargin}{-69pt} \begin{document} \begin{equation*}{\mathrm{_{B}^{-}}}\end{equation*}\end{document} curves. Moreover, notable increase of H+/Q\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \setlength{\oddsidemargin}{-69pt} \begin{document} \begin{equation*}{\mathrm{_{B}^{-}}}\end{equation*}\end{document} is observed in the L209 mutants at neutral pH as compared with the WT. The large effects observed in all L209 mutants are not associated with significant structural changes (Kuglstatter, A., Ermler, U., Michel, H., Baciou, L. & Fritzsch, G. Biochemistry (2001) 40, 4253–4260). Our data suggest that, in the L209 mutants, the QB cluster does not respond to the Q\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \setlength{\oddsidemargin}{-69pt} \begin{document} \begin{equation*}{\mathrm{_{A}^{-}}}\end{equation*}\end{document} formation as observed in the WT. We propose that, in the mutants, removal of the rigid proline L209 breaks a necessary hydrogen bonding connection between the quinone sites. These findings suggest an important role for structural rigidity in ensuring a functional interaction between quinone binding sites.

The cytosolic subunit p67phox of the NADPH-oxidase complex does not bind NADPH

The NADPH‐oxidase of phagocytic cells is a multicomponent enzyme that generates superoxide. It comprises a membrane flavocytochrome b 558 and four cytosolic proteins; p67phox, p47phox, p40phox and Rac. The NADPH‐binding site of this complex was shown to be located on the flavocytochrome b 558. However, a number of studies have suggested the presence of another site on the p67phox subunit which is the key activating component. Using several approaches like tryptophan quenching fluorescence measurement, inhibition by 2′,3′‐dialdehyde NADPH, and free/bound NADPH concentration measurements, we demonstrate that no NADPH binds on p67phox, thus definitively solving the controversy on the number and location of the NADPH‐binding sites on this complex.

Titanium Dioxide Nanoparticles Increase Superoxide Anion Production by Acting on NADPH Oxidase

Titanium dioxide (TiO2) anatase nanoparticles (NPs) are metal oxide NPs commercialized for several uses of everyday life. However their toxicity has been poorly investigated. Cellular internalization of NPs has been shown to activate macrophages and neutrophils that contribute to superoxide anion production by the NADPH oxidase complex. Transmission electron micrososcopy images showed that the membrane fractions were close to the NPs while fluorescence indicated an interaction between NPs and cytosolic proteins. Using a cell-free system, we have investigated the influence of TiO2 NPs on the behavior of the NADPH oxidase. In the absence of the classical activator molecules of the enzyme (arachidonic acid) but in the presence of TiO2 NPs, no production of superoxide ions could be detected indicating that TiO2 NPs were unable to activate by themselves the complex. However once the NADPH oxidase was activated (i.e., by arachidonic acid), the rate of superoxide anion production went up to 140% of its value without NPs, this effect being dependent on their concentration. In the presence of TiO2 nanoparticles, the NADPH oxidase produces more superoxide ions, hence induces higher oxidative stress. This hyper-activation and the subsequent increase in ROS production by TiO2 NPs could participate to the oxidative stress development.

The DMPC lipid phase transition influences differently the first and the second electron transfer reactions in bacterial reaction centers

Photosynthetic reaction centers (RCs) from Rhodobacter sphaeroides were incorporated in dimyristoylphosphatidylcholine (DMPC) liposomes. The first and second electron transfer rates (k AB(1) and k AB(2), respectively) between the first and the second quinone electron acceptors have been measured as a function of temperature, across the phase transition of DMPC (23°C). The Eyring plots of k AB(1) display straight lines. In contrast, the Eyring plots for k AB(2) in proteoliposomes show a break at about 23.5°C. This physical discrimination between the two electron transfer reactions demonstrates that the stiffness of the lipid environment of the RCs and/or the protein–protein interactions influence the parameters governing k AB(2), but not the gating process limiting k AB(1).

HETEROGENEITY OF THE QUINONE ELECTRON ACCEPTOR SYSTEM IN BACTERIAL REACTION CENTERS

Abstract— In reaction centers from Rhodopseudomonas viridis, biphasicity of the charge recombination kinetics between P+, the primary electron donor, and QA and QB‐, the primary and secondary quinone electron acceptors, respectively, have been analyzed by the flash‐induced absorption change technique. We have studied the effect of quinone environment modifications on the ratio of the two phases for the P+QA‐ ([Afast/Aslow]a) and P+QB‐ ([Afast/Aslow]b) charge recombination processes. In reaction centers from Rps. viridis reconstituted in phosphatidylcholine liposomes a notable influence of the nature of the QB pocket occupancy was observed on (Afast/Aslow)a. This ratio is much affected by the presence of o‐phenanthroline compared to reaction centers with an empty QB pocket or with terbutryn present. Because o‐phenanthroline was proposed to hydrogen bind HisL190, whereas terbutryn does not, we suggest that a HisLI90‐Fe‐HisM217 (the equivalent to HisLI90 in the QA pocket) “wire” may be involved in the existence of the two conformational states associated with the two phases of charge recombination. In chromat‐ophores from the T1 (SerL223→ Ala; ArgL217→ His) and T4 (TyrL222→ Phe) mutants no modification of the (Afast/Aslow)a ratio is detected, whereas the (Afast/Aslow)b ratios are substantially modified compared to the wild type (WT). In the T3 mutant (PheL216→ Ser; ValM263→ Phe [4.1 Å apart from QA]), (Afast/Aslow)a is notably changed compared to the WT. Our data show that any modification in the close protein environment of the quinones and/or of the HisL190 and HisM217 affects the equilibrium between the two reaction center states. This is consistent with the existence of two reaction center states from Rps. viridis, associated with two different conformations of the quinones‐histidines‐iron system. This “wire” allows both quinone protein pockets to interact over quite long distances.

Are the Fluorescent Properties of the Cyan Fluorescent Protein Sensitive to Conditions of Oxidative Stress

The modifications induced by reactive oxygen species (ROS) on fluorescent proteins (FPs) may have important implications for live cell fluorescence imaging. Using quantitative γ‐radiolysis, we have studied the ROS‐induced biochemical and photophysical perturbations on recombinant cyan fluorescent protein (CFP). After oxidation by the ˙OH radical, the protein displays a modified RP‐HPLC elution profile, while the CFP fluorescence undergoes pronounced decreases in intensity and lifetime, without changes in its excitation and emission spectra. Meanwhile, the Förster resonant energy transfer (FRET) between the single W57 and the chromophore remains unperturbed. These results rule out a direct oxidation of the CFP chromophore and of W57 as well as major changes in the protein 3D structure, but show that new fluorescent forms associated to a higher level of dynamic quenching have been generated. Thus, strict in situ controls are required when CFP is to be used for FRET studies in situations of oxidative activity, or under strong illumination.

Assembly of phagocyte NADPH oxidase: A concerted binding process?

BACKGROUND The phagocyte NADPH-oxidase is a multicomponent enzyme that generates superoxide anions. It comprises a membrane redox component flavocytochrome b558 and four cytosolic proteins (p67(phox), p47(phox), p40(phox) and Rac) that must assemble to produce an active system. In this work we focused on the spatio-temporal control of the activation process of phagocyte NADPH oxidase. METHODS A wide range of techniques including fast kinetics with a stopped-flow apparatus and various combinations of the activating factors was used to test the order of assembly and the role of the p47(phox)-p67(phox) complex. RESULTS The data presented here are consistent with the absence of a catalytic role of the p47(phox)-p67(phox) interacting state and support the idea of independent binding sites for the cytosolic proteins on the flavocytochrome b558 allowing random binding order. However, the formation of the active complex appears to involve a synergistic process of binding of the activated cytosolic subunits to cytochrome b558. All partners should be in the vicinity for optimal assembly, a delay or the absence of one of the partners in this process seems to lead to a decrease in the efficiency of the catalytic core. CONCLUSION AND GENERAL SIGNIFICANCE The activation and assembly of the NADPH oxidase components have to be achieved simultaneously for the formation of an efficient and optimal enzyme complex. This mechanism appears to be incompatible with continuous fast exchanges of the cytosolic proteins during the production of superoxide ion in the phagosome.