Role of RhdA in GS^•-scavenging.

<p>The scheme displays a possible interplay of RhdA-catalyzed GS^•-scavenging (thick lines) with other players (grey lines) involved in GSH homeostasis. The oxygen-dependent GS^•-scavenging activity of RhdA would allow the dissipation of GS^• as disulfide glutathione (GSSG), and GSH is produced from GSSG by the glutathione reductase activity (GR). The superoxide anion can be removed by the generally recognized players in superoxide scavenging machinery (i.e. superoxide dismutase, SOD, and catalase, KAT). The inset depicts the likely <b>feature</b> of the RhdA active-site at the beginning of the GS^•-scavenging reaction.</p

<i>In silico</i> docking of glutathione and thiosulfate to RhdA.

<p>Overall RhdA structure (<b>A</b>) and its active site region interacting with either reduced glutathione (<b>B</b>) and thiosulfate (<b>C</b>). The amino acidic residues (Arg²³⁵, Thr²³² and Cys²³⁰) involved in the interactions are highlighted in black. Ligand backbones are displayed as dark grey sticks. <i>In silico</i> computed hydrogen bonds between ligands and amino acidic residues are displayed as light grey solid lines. Images of molecular docking has been generated using UCSF chimera software (<a href="http://www.cgl.ucsf.edu/chimera/" target="_blank">http://www.cgl.ucsf.edu/chimera/</a>).</p

Glutathione in <i>A. vinelandii</i>.

<p>(<b>A</b>) Glutathione was detected in wild-type (UW136; white bars) and in RhdA-null mutant (MV474; grey bars) <i>A. vinelandii</i> strains grown in Burk's medium containing sucrose (BSN) or gluconate (BGN) as carbon source in the absence (not-dotted bars) and in the presence (dotted bars) of phenazine methosulfate (PMS). Measured glutathione includes both the total reduced-glutathione and the DTT-reducible fraction of total free glutathione and is expressed as function of the protein amount detected in 10 mM Tris-HCl 0.1 M NaCl (pH 8) extracts of cell-samples from the same growths. All data are the mean of three independent replicates ± standard deviation. Differences significant for <i>P</i><0.01 (**) are indicated (Student's <i>t</i> test).</p

Intrinsic fluorescence changes of RhdA and RhdA-Cys²³⁰Ala in the presence of GSH.

<p>Tryptophan fluorescence emission spectra (λ_ex = 280 nm; dotted lines) of RhdA (<b>A</b>) and RhdA-Cys²³⁰Ala (<b>B</b>) in the presence of 50 µM GSH. Solid lines report spectra before the addition of GSH. Both RhdA and RhdA-Cys²³⁰Ala were 2 µM in 50 mM Tris-HCl, 100 mM NaCl (pH 7.4). Spectra were corrected for dilution.</p

Changes of the intrinsic fluorescence of RhdA upon titration with glutathione species.

<p>The amounts indicated in abscissa of GSH (<b>A</b>, full circles), GS^• (<b>A</b>, empty circles), and GSSG (<b>B</b>, full circles) were successively added to 2 µM RhdA in 50 mM Tris-HCl, 100 mM NaCl (pH 7.4). Fluorescence intensity was measured at 340 nm (λ_ex = 280 nm), and was corrected for dilution.</p

Iron Binding Properties of Recombinant Class A Protein Disulfide Isomerase from <i>Arabidopsis thaliana</i>

The protein disulfide isomerase (PDI) family comprises a wide set of enzymes mainly involved in thiol–disulfide exchange reactions in the endoplasmic reticulum. Class A PDIs (PDI-A) constitute the smallest members of the family, consisting of a single thioredoxin (TRX) module without any additional domains. To date, their catalytic activity and cellular function are still poorly understood. To gain insight into the role of higher-plant class A PDIs, the biochemical properties of r<i>At</i>PDI-A, the recombinant form of <i>Arabidopsis thaliana</i> PDI-A, have been investigated. As expressed, r<i>At</i>PDI-A has only little oxidoreductase activity, but it appears to be capable of binding an iron–sulfur (Fe–S) cluster, most likely a [2Fe-2S] center, at the interface between two protein monomers. A mutational survey of all cysteine residues of r<i>At</i>PDI-A indicates that only the second and third cysteines of the CXX­X­C­KHC stretch, containing the putative catalytic site CKHC, are primarily involved in cluster coordination. A key role is also played by the lysine residue. Its substitution with glycine, which restores the canonical PDI active site CGHC, does not influence the oxidoreductase activity of the protein, which remains marginal, but strongly affects the binding of the cluster. It is therefore proposed that the unexpected ability of r<i>At</i>PDI-A to accommodate an Fe–S cluster is due to its very unique CKHC motif, which is conserved in all higher-plant class A PDIs, differentiating them from all other members of the PDI family

Analyses of the RhdA/GSH interaction.

<p>(<b>A</b>) Thermally-induced fluorescence changes of 2 µM RhdA (solid line) and RhdA/GSH complex (dotted line) in 50 mM Tris-HCl, 100 mM NaCl (pH 7.4). Tryptophan fluorescence was monitored as a function of the indicated temperature. Transition temperatures of the first conformational change are indicated. (<b>B</b>) Tryptophan fluorescence emission spectra (λ_ex = 280 nm) of 2 µM RhdA/GSH complex alone (dotted line) in 50 mM Tris-HCl, 100 mM NaCl (pH 7.4), in the presence of 0.8 mM Tris(2-carboxyethyl)phosphine (TCEP; long-dashed line), and in the presence of 0.2 mM DTT (short-dashed line). For RhdA/GSH complex preparation, RhdA was treated with GSH and unbound GSH was removed by gel-filtration. Solid line reports the spectrum of 2 µM RhdA alone. (<b>C</b>) Tryptophan fluorescence emission spectra (λ_ex = 280 nm) of 2 µM RhdA alone in high-saline concentrated buffer (50 mM Tris-HCl, 1 M NaCl) (solid line) and in the presence of 0.25 mM GSH (dotted line). Spectrum of 2 µM RhdA in the presence 0.25 mM thiosulfate is reported as a positive control (dashed line). (<b>D</b>) LC/ESI Q-ToF analysis of RhdA/GSH complex prepared by 20 min incubation (25°C) of RhdA with 8-fold molar excess GSH, followed by desalting on C4-chromatographic pipet tip. Molecular mass range of the deconvoluted ESI spectra of RhdA (grey line) and RhdA/GSH complex (black line) is shown.</p

Trapping Dynamics in Photosystem I‑Light Harvesting Complex I of Higher Plants Is Governed by the Competition Between Excited State Diffusion from Low Energy States and Photochemical Charge Separation

The dynamics of excited state equilibration and primary photochemical trapping have been investigated in the photosystem I-light harvesting complex I isolated from spinach, by the complementary time-resolved fluorescence and transient absorption approaches. The combined analysis of the experimental data indicates that the excited state decay is described by lifetimes in the ranges of 12–16 ps, 32–36 ps, and 64–77 ps, for both detection methods, whereas faster components, having lifetimes of 550–780 fs and 4.2–5.2 ps, are resolved only by transient absorption. A unified model capable of describing both the fluorescence and the absorption dynamics has been developed. From this model it appears that the majority of excited state equilibration between the bulk of the antenna pigments and the reaction center occurs in less than 2 ps, that the primary charge separated state is populated in ∼4 ps, and that the charge stabilization by electron transfer is completed in ∼70 ps. Energy equilibration dynamics associated with the long wavelength absorbing/emitting forms harbored by the PSI external antenna are also characterized by a time mean lifetime of ∼75 ps, thus overlapping with radical pair charge stabilization reactions. Even in the presence of a kinetic bottleneck for energy equilibration, the excited state dynamics are shown to be principally trap-limited. However, direct excitation of the low energy chlorophyll forms is predicted to lengthen significantly (∼2-folds) the average trapping time