Francesco Francia

Redox regulation of the Calvin–Benson cycle: something old, something new

Reversible redox post-translational modifications such as oxido-reduction of disulfide bonds, S-nitrosylation, and S-glutathionylation, play a prominent role in the regulation of cell metabolism and signaling in all organisms. These modifications are mainly controlled by members of the thioredoxin and glutaredoxin families. Early studies in photosynthetic organisms have identified the Calvin–Benson cycle, the photosynthetic pathway responsible for carbon assimilation, as a redox regulated process. Indeed, 4 out of 11 enzymes of the cycle were shown to have a low activity in the dark and to be activated in the light through thioredoxin-dependent reduction of regulatory disulfide bonds. The underlying molecular mechanisms were extensively studied at the biochemical and structural level. Unexpectedly, recent biochemical and proteomic studies have suggested that all enzymes of the cycle and several associated regulatory proteins may undergo redox regulation through multiple redox post-translational modifications including glutathionylation and nitrosylation. The aim of this review is to detail the well-established mechanisms of redox regulation of Calvin–Benson cycle enzymes as well as the most recent reports indicating that this pathway is tightly controlled by multiple interconnected redox post-translational modifications. This redox control is likely allowing fine tuning of the Calvin–Benson cycle required for adaptation to varying environmental conditions, especially during responses to biotic and abiotic stresses.

Reduction of potassium tellurite to elemental tellurium and its effect on the plasma membrane redox components of the facultative phototroph Rhodobacter capsulatus.

Summary.u2002Anaerobically light-grown cells of Rhodobacter capsulatus B100 are highly resistant to the toxic oxyanion tellurite (TeO32−; minimal inhibitory concentration, 250u2009μg/ml). This study examines, for the first time, some structural and biochemical features of cells and plasma membrane fragments of this facultative phototroph grown in the presence of 50μg of K2TeO3 per ml. Through the use of transmission microscopy and X-ray microanalysis we show that several “needlelike” shaped granules of elemental tellurium are accumulated into the cytosol near the intracytoplasmic membrane system. Flash-spectroscopy, oxygen consumption measurements, and difference spectra analysis indicated that membrane vesicles (chromatophores) isolated from tellurite-grown cells are able to catalyze both photosynthetic and respiratory electron transport activities, although they are characterized by a low c-type cytochrome content (mostly soluble cytochrome c2). This feature is paralleled by a low cytochrome c oxidase activity and with an NADH-dependent respiration which is catalyzed by a pathway leading to a quinol oxidase (Qox) inhibited by high (millimolar) concentrations of cyanide (CN−). Conversely, membranes from R. capsulatus B100 cells grown in the absence of tellurite are characterized by a branched respiratory chain in which the cytochrome c oxidase pathway (blocked by CN− in the micromolar range) accounts for 35–40% of the total NADH-dependent oxygen consumption, while the remaining activity is catalyzed by the quinol oxidase pathway. These data have been interpreted to show that tellurite resistance of R. capsulatus B100 is characterized by the presence of a modified plasma-membrane-associated electron transport system.

The Thiol:Disulfide Oxidoreductase DsbB Mediates the Oxidizing Effects of the Toxic Metalloid Tellurite (TeO32−) on the Plasma Membrane Redox System of the Facultative Phototroph Rhodobacter capsulatus

The highly toxic oxyanion tellurite (TeO3(2-)) is a well known pro-oxidant in mammalian and bacterial cells. This work examines the effects of tellurite on the redox state of the electron transport chain of the facultative phototroph Rhodobacter capsulatus, in relation to the role of the thiol:disulfide oxidoreductase DsbB. Under steady-state respiration, the addition of tellurite (2.5 mM) to membrane fragments generated an extrareduction of the cytochrome pool (c- and b-type hemes); further, in plasma membranes exposed to tellurite (0.25 to 2.5 mM) and subjected to a series of flashes of light, the rate of the QH2:cytochrome c (Cyt c) oxidoreductase activity was enhanced. The effect of tellurite was blocked by the antibiotics antimycin A and/or myxothiazol, specific inhibitors of the QH2:Cyt c oxidoreductase, and, most interestingly, the membrane-associated thiol:disulfide oxidoreductase DsbB was required to mediate the redox unbalance produced by the oxyanion. Indeed, this phenomenon was absent from R. capsulatus MD22, a DsbB-deficient mutant, whereas the tellurite effect was present in membranes from MD22/pDsbB(WT), in which the mutant gene was complemented to regain the wild-type DsbB phenotype. These findings were taken as evidence that the membrane-bound thiol:disulfide oxidoreductase DsbB acts as an electron conduit between the hydrophilic metalloid and the lipid-embedded Q pool, so that in habitats contaminated with subinhibitory amounts of Te(IV), the metalloid is likely to function as a disposal for the excess reducing power at the Q-pool level of facultative phototrophic bacteria.

Reduction of chalcogen oxyanions and generation of nanoprecipitates by the photosynthetic bacterium Rhodobacter capsulatus

The facultative photosynthetic bacterium Rhodobacter capsulatus is characterized in its interaction with the toxic oxyanions tellurite (Te(IV)) and selenite (Se(IV)) by a highly variable level of resistance that is dependent on the growth mode making this bacterium an ideal organism for the study of the microbial interaction with chalcogens. As we have reported in the past, while the oxyanion tellurite is taken up by R. capsulatus cells via acetate permease and it is reduced to Te(0) in the cytoplasm in the form of splinter-like black intracellular deposits no clear mechanism was described for Se(0) precipitation. Here, we present the first report on the biotransformation of tellurium and selenium oxyanions into extracellular Te(0) and Se(0)nanoprecipitates (NPs) by anaerobic photosynthetically growing cultures of R. capsulatus as a function of exogenously added redox-mediator lawsone, i.e. 2-hydroxy-1,4-naphthoquinone. The NPs formation was dependent on the carbon source used for the bacterial growth and the rate of chalcogen reduction was constant at different lawsone concentrations, in line with a catalytic role for the redox mediator. X-ray diffraction (XRD) analysis demonstrated the Te(0) and Se(0) nature of the nanoparticles.

Effects of dehydration on light-induced conformational changes in bacterial photosynthetic reaction centers probed by optical and differential FTIR spectroscopy.

Following light-induced electron transfer between the primary donor (P) and quinone acceptor (Q(A)) the bacterial photosynthetic reaction center (RC) undergoes conformational relaxations which stabilize the primary charge separated state P(+)Q(A)(-). Dehydration of RCs from Rhodobacter sphaeroides hinders these conformational dynamics, leading to acceleration of P(+)Q(A)(-) recombination kinetics [Malferrari et al., J. Phys. Chem. B 115 (2011) 14732-14750]. To clarify the structural basis of the conformational relaxations and the involvement of bound water molecules, we analyzed light-induced P(+)Q(A)(-)/PQ(A) difference FTIR spectra of RC films at two hydration levels (relative humidity r=76% and r=11%). Dehydration reduced the amplitude of bands in the 3700-3550cm(-1) region, attributed to water molecules hydrogen bonded to the RC, previously proposed to stabilize the charge separation by dielectric screening [Iwata et al., Biochemistry 48 (2009) 1220-1229]. Other features of the FTIR difference spectrum were affected by partial depletion of the hydration shell (r=11%), including contributions from modes of P (9-keto groups), and from NH or OH stretching modes of amino acidic residues, absorbing in the 3550-3150cm(-1) range, a region so far not examined in detail for bacterial RCs. To probe in parallel the effects of dehydration on the RC conformational relaxations, we analyzed by optical absorption spectroscopy the kinetics of P(+)Q(A)(-) recombination following the same photoexcitation used in FTIR measurements (20s continuous illumination). The results suggest a correlation between the observed FTIR spectral changes and the conformational rearrangements which, in the hydrated system, strongly stabilize the P(+)Q(A)(-) charge separated state over the second time scale.

Retardation of Protein Dynamics by Trehalose in Dehydrated Systems of Photosynthetic Reaction Centers. Insights from Electron Transfer and Thermal Denaturation Kinetics

Conformational protein dynamics is known to be hampered in amorphous matrixes upon dehydration, both in the absence and in the presence of glass forming disaccharides, like trehalose, resulting in enhanced protein thermal stability. To shed light on such matrix effects, we have compared the retardation of protein dynamics in photosynthetic bacterial reaction centers (RC) dehydrated at controlled relative humidity in the absence (RC films) or in the presence of trehalose (RC-trehalose glasses). Small scale RC dynamics, associated with the relaxation from the dark-adapted to the light-adapted conformation, have been probed up to the second time scale by analyzing the kinetics of electron transfer from the photoreduced quinone acceptor (QA(-)) to the photoxidized primary donor (P(+)) as a function of the duration of photoexcitation from 7 ns (laser pulse) to 20 s. A more severe inhibition of dynamics is found in RC-trehalose glasses than in RC films: only in the latter system does a complete relaxation to the light-adapted conformation occur even at extreme dehydration, although strongly retarded. To gain insight into the large scale RC dynamics up to the time scale of days, the kinetics of thermal denaturation have been studied at 44 °C by spectral analysis of the Qx and Qy bands of the RC bacteriochlorin cofactors, as a function of the sugar/protein molar ratio, m, varied between 0 and 10(4). Upon increasing m, denaturation is slowed progressively, and above m ∼ 500 the RC is stable at least for several days. The stronger retardation of RC relaxation and dynamics induced by trehalose is discussed in the light of a recent molecular dynamics simulation study performed in matrixes of the model protein lysozyme with and without trehalose. We suggest that the efficiency of trehalose in retarding RC dynamics and preventing thermal denaturation stems mainly from its propensity to form and stabilize extended networks of hydrogen bonds involving sugar, residual water, and surface residues of the RC complex and from its ability of reducing the free volume fraction of protein alone matrixes.

Trehalose Preserves the Integrity of Lyophilized Phycoerythrin–AntiHuman CD8 Antibody Conjugates and Enhances their Thermal Stability in Flow Cytometric Assays

An increasing number of publications report on the efficacy of trehalose in preserving organisms, cells, and macromolecules from adverse environmental conditions such as extreme temperatures and dryness. Although the mechanism by which this disaccharide exerts its protection is still debated, the implementation of trehalose as stabilizer is becoming a praxis in several preparative protocols from the pharmaceutical industry. We tested the ability of trehalose in protecting R-Phycoerythrin (R-PE), a pigment-protein complex widely used as fluorescent marker, from thermal denaturation. Once embedded into a dried trehalose matrix, R-PE retains its optical absorption-emission characteristics even when exposed to 70°C for h or when subjected to freeze-drying. We subsequently examined the protection exerted by trehalose on freeze-dried antihuman CD8-RPE (CD8-RPE) conjugated antibodies. Flow cytometric analysis showed that colyophilized trehalose-CD8-RPE preparations can be exposed for 4 weeks at 45°C without significant loss of functionality. Remarkably, even following 4 weeks incubation at 70°C, the preparations are still able to specifically recognize CD8(+) lymphocyte populations. These results show that colyophilization with trehalose makes possible the preparation of antibody-based diagnostic kits which can withstand breaks in the cold chain distribution, particularly suited for use in less-developed countries of the tropical areas.

A New Method for D2O/H2O Exchange in Infrared Spectroscopy of Proteins

In this paper, we describe a new method to obtain D2O/H2O exchange in photosynthetic reaction centres from Rhodobacter sphaeroides. The method is characterized by: (i) a very high efficiency of the isotopic replacement; (ii) an extremely low amount of D2O needed; (iii) the short time required for dehydration and D2O rehydration; (iv) the possibility of controlling concomitantly the hydration state of the sample. The proposed method can be applied to other proteins.

Dehydration affects the electronic structure of the primary electron donor in bacterial photosynthetic reaction centers: evidence from visible-NIR and light-induced difference FTIR spectroscopy.

The photosynthetic reaction center (RC) is a membrane pigment-protein complex that catalyzes the initial charge separation reactions of photosynthesis. Following photoexcitation, the RC undergoes conformational relaxations which stabilize the charge-separated state. Dehydration of the complex inhibits its conformational dynamics, providing a useful tool to gain insights into the relaxational processes. We analyzed the effects of dehydration on the electronic structure of the primary electron donor P, as probed by visible-NIR and light-induced FTIR difference spectroscopy, in RC films equilibrated at different relative humidities r. Previous FTIR and ENDOR spectroscopic studies revealed that P, an excitonically coupled dimer of bacteriochlorophylls, can be switched between two conformations, P866 and P850, which differ in the extent of delocalization of the unpaired electron between the two bacteriochlorophyll moieties (PL and PM) of the photo-oxidized radical P(+). We found that dehydration (at r = 11%) shifts the optical Qy band of P from 866 to 850-845 nm, a large part of the effect occurring already at r = 76%. Such a dehydration weakens light-induced difference FTIR marker bands, which probe the delocalization of charge distribution within the P(+) dimer (the electronic band of P(+) at 2700 cm(-1), and the associated phase-phonon vibrational modes at around 1300, 1480, and 1550 cm(-1)). From the analysis of the P(+) keto C[double bond, length as m-dash]O bands at 1703 and 1713-15 cm(-1), we inferred that dehydration induces a stronger localization of the unpaired electron on PL(+). The observed charge redistribution is discussed in relation to the dielectric relaxation of the photoexcited RC on a long (10(2) s) time scale.

Structural basis for the magnesium-dependent activation of transketolase from Chlamydomonas reinhardtii

BACKGROUNDnIn photosynthetic organisms, transketolase (TK) is involved in the Calvin-Benson cycle and participates to the regeneration of ribulose-5-phosphate. Previous studies demonstrated that TK catalysis is strictly dependent on thiamine pyrophosphate (TPP) and divalent ions such as Mg2+.nnnMETHODSnTK from the unicellular green alga Chlamydomonas reinhardtii (CrTK) was recombinantly produced and purified to homogeneity. Biochemical properties of the CrTK enzyme were delineated by activity assays and its structural features determined by CD analysis and X-ray crystallography.nnnRESULTSnCrTK is homodimeric and its catalysis depends on the reconstitution of the holo-enzyme in the presence of both TPP and Mg2+. Activity measurements and CD analysis revealed that the formation of fully active holo-CrTK is Mg2+-dependent and proceeds with a slow kinetics. The 3D-structure of CrTK without cofactors (CrTKapo) shows that two portions of the active site are flexible and disordered while they adopt an ordered conformation in the holo-form. Oxidative treatments revealed that Mg2+ participates in the redox control of CrTK by changing its propensity to be inactivated by oxidation. Indeed, the activity of holo-form is unaffected by oxidation whereas CrTK in the apo-form or reconstituted with the sole TPP show a strong sensitivity to oxidative inactivation.nnnCONCLUSIONnThese evidences indicate that Mg2+ is fundamental to allow gradual conformational arrangements suited for optimal catalysis. Moreover, Mg2+ is involved in the control of redox sensitivity of CrTK.nnnGENERAL SIGNIFICANCEnThe importance of Mg2+ in the functionality and redox sensitivity of CrTK is correlated to light-dependent fluctuations of Mg2+ in chloroplasts.