Comparative excitation-emission dependence of the FV /FM ratio in model green algae and cyanobacterial strains.

The emission spectra collected under conditions of open (F0 ) and closed (FM ) photosystem II (PSII) reaction centres are close-to-independent from the excitation wavelength in Chlamydomonas reinhardtii and Chlorella sorokiniana, whereas a pronounced dependence is observed in Synechocystis sp. PCC6803 and Synechococcus PCC7942, instead. The differences in band-shape between the F0 and FM emission are limited in green algae, giving rise only to a minor trough in the FV /FM spectrum in the 705-720 nm range, irrespectively of the excitation. More substantial variations are observed in cyanobacteria, resulting in marked dependencies of the measured FV /FM ratios on both the excitation and the detection wavelengths. In cyanobacteria, the maximal FV /FM values (0.5-0.7), observed monitoring at approximately 684 nm and exciting Chl a preferentially, are comparable to those of green algae; however, FV /FM decreases sharply below approximately 660 nm. Furthermore, in the red emission tail, the trough in the FV /FM spectrum is more pronounced in cyanobacteria with respect to green algae, corresponding to FV /FM values of 0.25-0.4 in this spectral region. Upon direct phycobilisomes excitation (i.e. >520 nm), the FV /FM value detected at 684 nm decreases to 0.3-0.5 and is close-to-negligible (approximately 0.1) below 660 nm. At the same time, the FV spectra are, in all species investigated, almost independent on the excitation wavelength. It is concluded that the excitation/emission dependencies of the FV /FM ratio arise from overlapped contributions from the three independent emissions of PSI, PSII and a fraction of energetically uncoupled external antenna, excited in different proportions depending on the respective optical cross-section and fluorescence yield.

Iron Binding Properties of Recombinant Class A Protein Disulfide Isomerase from Arabidopsis thaliana.

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 rAtPDI-A, the recombinant form of Arabidopsis thaliana PDI-A, have been investigated. As expressed, rAtPDI-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 rAtPDI-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 rAtPDI-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.

Spectral dependence of irreversible light-induced fluorescence quenching: Chlorophyll forms with maximal emission at 700-702 and 705-710nm as spectroscopic markers of conformational changes in the core complex.

The spectral dependence of the irreversible non-photochemical fluorescence quenching associated with photoinhibition in vitro has been comparatively investigated in thylakoid membranes, PSII enriched particles and PSII core complexes isolated from spinach. The analysis of the fluorescence emission spectra of dark-adapted and quenched samples as a function of the detection temperature in the 280-80K interval, indicates that Chlorophyll spectral forms having maximal emission in the 700-702nm and 705-710nm ranges gain relative intensity in concomitance with the establishment of irreversible light-induced quenching, acting thereby as spectroscopic markers. The relative enhancement of the 700-702nm and 705-710nm forms emission could be due either to an increase of their stoichiometric abundance or to their intrinsically low fluorescence quantum yields. These two factors, that can also coexist, need to be promoted by light-induced alterations in chromophore-protein as well as chromophore-chromophore interactions. The bands centred at about 701 and 706nm are also observed in the PSII core complex, suggesting their, at least partial, localisation in proximity to the reaction centre, and the occurrence of light-induced conformational changes in the core subunits.

Kinetics and heterogeneity of energy transfer from light harvesting complex II to photosystem I in the supercomplex isolated from Arabidopsis.

State transitions are a phenomenon that maintains the excitation balance between photosystem II (PSII) and photosystem I (PSI-LHCI) by controlling their relative absorption cross-sections. Under light conditions exciting PSII preferentially, a trimeric LHCII antenna moves from PSII to PSI-LHCI to form the PSI-LHCI-LHCII supercomplex. In this work, the excited state dynamics in the PSI-LHCI and PSI-LHCI-LHCII supercomplexes isolated from Arabidopsis have been investigated by picosecond time-resolved fluorescence spectroscopy. The excited state decays were analysed using two approaches based on either (i) a sum of discrete exponentials or (ii) a continuous distribution of lifetimes. The results indicate that the energy transfer from LHCII to the bulk of the PSI antenna occurs with an average macroscopic transfer rate in the 35-65 ns-1 interval. Yet, the most satisfactory description of the data is obtained when considering a heterogeneous population containing two PSI-LHCI-LHCII supercomplexes characterised by a transfer time of ∼15 and ∼60 ns-1, likely due to the differences in the strength and orientation of LHCII harboured to PSI. Both these values are of the same order of magnitude of those estimated for the average energy transfer rates from the low energy spectral forms of LHCI to the bulk of the PSI antenna (15-40 ns-1), but they are slower than the transfer from the bulk antenna of PSI to the reaction centre (>150 ns-1), implying a relatively small kinetics bottleneck for the energy transfer from LHCII. Nevertheless, the kinetic limitation imposed by excited state diffusion has a negligible impact on the photochemical quantum efficiency of the supercomplex, which remains about 98% in the case of PSI-LHCI.

Altered expression level of Escherichia coli proteins in response to treatment with the antifouling agent zosteric acid sodium salt.

Zosteric acid sodium salt is a powerful antifouling agent. However, the mode of its antifouling action has not yet been fully elucidated. Whole cell proteome of Escherichia coli was analysed to study the different protein patterns expressed by the surface-exposed planktonic cells without and with sublethal concentrations of the zosteric acid sodium salt. Proteomic analysis revealed that at least 27 proteins showed a significant (19 upregulated and 8 downregulated, P < 0.001) altered expression level in response to the antifoulant. The proteomic signatures of zosteric acid sodium salt-treated cells are characterized by stress-associated (e.g. AhpC, OsmC, SodB, GroES, IscU, DnaK), motility-related (FliC), quorum-sensing-associated (LuxS) and metabolism/biosynthesis-related (e.g. PptA, AroA, FabD, FabB, GapA) proteins. Consistent with the overexpression of LuxS enzyme, the antifouling agent increased autoinducer-2 (AI-2) concentration by twofold. Moreover, treated cells experienced a statistically significant but modest increase of reactive oxygen species (+ 23%), tryptophanase (1.2-fold) and indole (1.2-fold) synthesis. Overall, our data suggest that zosteric acid sodium salt acts as environmental cue leading to global stress on E. coli cells, which favours the expression of various protective proteins, the AI-2 production and the synthesis of flagella, to escape from adverse conditions.

Effects of chronic sub-lethal oxidative stress on biofilm formation by Azotobacter vinelandii.

This work showed that perturbations of the physiological steady-state level of reactive oxygen species (ROS) affected biofilm genesis and the characteristics of the model bacterium Azotobacter vinelandii. To get a continuous endogenous source of ROS, a strain exposed to chronic sub-lethal oxidative stress was deprived of the gene coding for the antioxidant rhodanese-like protein RhdA (MV474). In this study MV474 biofilm showed (i) a seven-fold higher growth rate, (ii) induction of catalase and alkyl-hydroxyl-peroxidase enzymes, (iii) higher average thicknesses due to increased production of a polysaccharide-rich extracellular matrix and (iv) less susceptibility to hydrogen peroxide than the wild-type strain (UW136). MV474 showed increased swimming and swarming activity and the swarming colonies experienced a higher level of oxidative stress compared to UW136. A continuous exogenous source of ROS increased biofilm formation in UW136. Overall, chronic sub-lethal oxidative events promoted sessile behavior in A. vinelandii.

Mobilization of sulfane sulfur from cysteine desulfurases to the Azotobacter vinelandii sulfurtransferase RhdA.

Mobilization of the L-cysteine sulfur for the persulfuration of the rhodanese of Azotobacter vinelandii, RhdA, can be mediated by the A. vinelandii cysteine desulfurases, IscS and NifS. The amount of cysteine was higher in mutant strains lacking rhdA (MV474) than in wild type. The diazotrophic growth of MV474 was impaired. Taking into account the functional results about rhodanese-like proteins and RhdA itself, it is suggested that RhdA-dependent modulation of L-cysteine levels must deal with a redox-related process.

The rhodanese RhdA helps Azotobacter vinelandii in maintaining cellular redox balance.

The tandem domain rhodanese-homology protein RhdA of Azotobacter vinelandii shows an active-site loop structure that confers structural peculiarity in the environment of its catalytic cysteine residue. The in vivo effects of the lack of RhdA were investigated using an A. vinelandii mutant strain (MV474) in which the rhdA gene was disrupted by deletion. Here, by combining analytical measurements and transcript profiles, we show that deletion of the rhdA gene generates an oxidative stress condition to which A. vinelandii responds by activating defensive mechanisms. In conditions of growth in the presence of the superoxide generator phenazine methosulfate, a stressor-dependent induction of rhdA gene expression was observed, thus highlighting that RhdA is important for A. vinelandii to sustain oxidative stress. The potential of RhdA to buffer general levels of oxidants in A. vinelandii cells via redox reactions involving its cysteine thiol is discussed.

Excitation and emission wavelength dependence of fluorescence spectra in whole cells of the cyanobacterium Synechocystis sp. PPC6803: Influence on the estimation of Photosystem II maximal quantum efficiency.

The fluorescence emission spectrum of Synechocystis sp. PPC6803 cells, at room temperature, displays: i) significant bandshape variations when collected under open (F0) and closed (FM) Photosystem II reaction centre conditions; ii) a marked dependence on the excitation wavelength both under F0 and FM conditions, due to the enhancement of phycobilisomes (PBS) emission upon their direct excitation. As a consequence: iii) the ratio of the variable and maximal fluorescence (FV/FM), that is a commonly employed indicator of the maximal photochemical quantum efficiency of PSII (Φpc, PSII), displays a significant dependency on both the excitation and the emission (detection) wavelength; iv) the FV/FM excitation/emission wavelength dependency is due, primarily, to the overlap of PSII emission with that of supercomplexes showing negligible changes in quantum yield upon trap closure, i.e. PSI and a PBS fraction which is incapable to transfer the excitation energy efficiently to core complexes. v) The contribution to the cellular emission and the relative absorption-cross section of PSII, PSI and uncoupled PBS are extracted using a spectral decomposition strategy. It is concluded that vi) Φpc, PSII is generally underestimated from the FV/FM measurements in this organism and, the degree of the estimation bias, which can exceed 50%, depends on the measurement conditions. Spectral modelling based on the decomposed emission/cross-section profiles were extended to other processes typically monitored from steady-state fluorescence measurements, in the presence of an actinic illumination, in particular non-photochemical quenching. It is suggested that vii) the quenching extent is generally underestimated in analogy to FV/FM but that viii) the location of quenching sites can be discriminated based on the combined excitation/emission spectral analysis.

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.

Two wavelength-dependent mechanisms of sensitisation of light-induced quenching in the isolated light-harvesting complex II.

The efficiency of visible light in inducing fluorescence quenching in the isolated light-harvesting complex II (LHCII) of higher plants is investigated by action spectroscopy in the visible portion of photosynthetic active radiation. The efficiency spectrum displays a relatively homogenous quenching yield across the most intense electronic transitions of the chlorophyll a and carotenoid pigments, indicating that quenching proceeds from the equilibrated state of the complex. Larger yields are observed in the 510-640-nm window, where weak transitions of LHCII-bound chromophores occur. This observation is interpreted in terms of an additional quenching sensitisation process mediated by these electronic transitions.

Involvement of the Azotobacter vinelandii rhodanese-like protein RhdA in the glutathione regeneration pathway.

The phenotypic features of the Azotobacter vinelandii RhdA mutant MV474 (in which the rhdA gene was deleted) indicated that defects in antioxidant systems in this organism were related to the expression of the tandem-domain rhodanese RhdA. In this work, further insights on the effects of the oxidative imbalance generated by the absence of RhdA (e.g. increased levels of lipid hydroperoxides) are provided. Starting from the evidence that glutathione was depleted in MV474, and using both in silico and in vitro approaches, here we studied the interaction of wild-type RhdA and Cys(230)Ala site-directed RhdA mutant with glutathione species. We found that RhdA was able to bind in vitro reduced glutathione (GSH) and that RhdA-Cys(230) residue was mandatory for the complex formation. RhdA catalyzed glutathione-disulfide formation in the presence of a system generating the glutathione thiyl radical (GS, an oxidized form of GSH), thereby facilitating GSH regeneration. This reaction was negligible when the Cys(230)Ala RhdA mutant was used. The efficiency of RhdA as catalyst in GS-scavenging activity is discussed on the basis of the measured parameters of both interaction with glutathione species and kinetic studies.