Donatella Carbonera

Permeability of inner mitochondrial membrane and oxidative stress

The mechanism of increase in the inner membrane permeability induced by Ca2+ plus Pi, diamide and hydroperoxides has been analyzed. (1) The permeability increase is antagonized by oligomycin and favoured by atractyloside. The promoting effect of atractyloside is strongly reduced if the mitochondria are simultaneously treated with oligomycin. (2) Addition of the free-radical scavenger, butylhydroxytoluene, results in a complete protection of the membrane with respect to the permeability increase. (3) Although membrane damage and depression of the GSH concentration are often associated, there is no direct correlation between extent of membrane damage and concentration of reduced glutathione. Abolition of the permeability increase by butylhydroxytoluene or by oligomycin is not accompanied by maintenance of a high GSH concentration in the presence of diamide or hydroperoxides. The membrane damage induced by Ca2+ plus Pi is not accompanied by a depression of the GSH concentration. (4) It is proposed that a variety of processes causing an increased permeability of the inner mitochondrial membrane merge into some ultimate common steps involving the action of oxygen radicals.

Microwave and optical spectroscopy of carotenoid triplets in light-harvesting complex LHC II of spinach by absorbance-detected magnetic resonance

Absorbance-detected magnetic resonance (ADMR) of the light-harvesting complex LHC II of spinach revealed two triplet contributions, having differentD values, but equalE value (|E|=0.00379 cm−1). The two triplets are assigned to two of the three carotenoids present in LHC II: lutein (|D|=0.03853 cm−1) and neoxanthin (|D|=0.04003 cm−1). The ADMR-detected Triplet-minus-Singlet (T—S) optical difference spectrum of the carotenoid (Car) triplet transition of LHC II showed, apart from bands in the Car absorption region, a contribution in the chlorophyll (Chl) absorption region due to a change in interaction between lutein and Chla at 670 nm, and neoxanthin and Chla at 670 and 677 nm. From Linear Dichroic (LD-)ADMR-detected LD-(T—S) spectra we have determined that the tripletz-axis (which corresponds roughly to the polyenal axis) of lutein and neoxanthin makes an angle of 47° and 38° with theQy transition moment of their adjacent Chla molecules, for the Chls absorbing at 670 and 677 nm, respectively. TheTz triplet magnetic transition moment of lutein is parallel to the lutein singlet and triplet absorptions, whereas theTx axis of neoxanthin makes an angle of about 20 degrees with the optical transition moments of the carotenoid molecule. The major Chla absorption bands of the optical absorption spectrum and the ADMR-detected T—S spectrum is best explained by assuming that all Chla is present in dimers. It is proposed that a free Chl dimer absorbs at 664 and 670 nm, whereas a Chl dimer bound to a carotenoid absorbs at 670 and 677 nm.

Zeaxanthin protects plant photosynthesis by modulating chlorophyll triplet yield in specific light-harvesting antenna subunits

Background: The plant carotenoid zeaxanthin is accumulated under excess light. Results: Zeaxanthin induces a red shift in the carotenoid triplet excited state spectrum and reveals a higher efficiency in controlling chlorophyll triplet formation. Conclusion: Binding of zeaxanthin to specific proteins modulates the yield of dangerous chlorophyll excited states and protects photosynthesis from over-excitation. Significance: Functional dissection of zeaxanthin-dependent photoprotective mechanisms is crucial for understanding how plants avoid photoinhibition. Plants are particularly prone to photo-oxidative damage caused by excess light. Photoprotection is essential for photosynthesis to proceed in oxygenic environments either by scavenging harmful reactive intermediates or preventing their accumulation to avoid photoinhibition. Carotenoids play a key role in protecting photosynthesis from the toxic effect of over-excitation; under excess light conditions, plants accumulate a specific carotenoid, zeaxanthin, that was shown to increase photoprotection. In this work we genetically dissected different components of zeaxanthin-dependent photoprotection. By using time-resolved differential spectroscopy in vivo, we identified a zeaxanthin-dependent optical signal characterized by a red shift in the carotenoid peak of the triplet-minus-singlet spectrum of leaves and pigment-binding proteins. By fractionating thylakoids into their component pigment binding complexes, the signal was found to originate from the monomeric Lhcb4–6 antenna components of Photosystem II and the Lhca1–4 subunits of Photosystem I. By analyzing mutants based on their sensitivity to excess light, the red-shifted triplet-minus-singlet signal was tightly correlated with photoprotection in the chloroplasts, suggesting the signal implies an increased efficiency of zeaxanthin in controlling chlorophyll triplet formation. Fluorescence-detected magnetic resonance analysis showed a decrease in the amplitude of signals assigned to chlorophyll triplets belonging to the monomeric antenna complexes of Photosystem II upon zeaxanthin binding; however, the amplitude of carotenoid triplet signal does not increase correspondingly. Results show that the high light-induced binding of zeaxanthin to specific proteins plays a major role in enhancing photoprotection by modulating the yield of potentially dangerous chlorophyll-excited states in vivo and preventing the production of singlet oxygen.

A well resolved ODMR triplet minus singlet spectrum of P680 from PSII particles

An ADMR T‐S spectrum of the primary donor (P680) of photosystem II (PSII) was obtained from anaerobically photoreduced particles. The spectrum is the best resolved obtained so far having a main bleaching band at 684 nm with a linewidth of only 100 cm−1. The view that this spectrum is produced by native homogeneous P680 unlike those obtained before is defended. A small bleaching observed at 678 nm is discussed in terms of the reaction center structure. One possible interpretation of the observations is that P680 is a very loose dimer with an exciton splitting of only 144 cm−1 corresponding to a dimer center‐to‐center distance of roughly 11.5 Å.

Pulse ENDOR and density functional theory on the peridinin triplet state involved in the photo-protective mechanism in the peridinin–chlorophyll a–protein from Amphidinium carterae

The photoexcited triplet state of the carotenoid peridinin in the Peridinin-chlorophyll a-protein of the dinoflagellate Amphidinium carterae has been investigated by pulse EPR and pulse ENDOR spectroscopies at variable temperatures. This is the first time that the ENDOR spectra of a carotenoid triplet in a naturally occurring light-harvesting complex, populated by energy transfer from the chlorophyll a triplet state, have been reported. From the electron spin echo experiments we have obtained the information on the electron spin polarization dynamics and from Mims ENDOR experiments we have derived the triplet state hyperfine couplings of the alpha- and beta-protons of the peridinin conjugated chain. Assignments of beta-protons belonging to two different methyl groups, with aiso=7.0 MHz and aiso=10.6 MHz respectively, have been made by comparison with the values predicted from density functional theory. Calculations provide a complete picture of the triplet spin density on the peridinin molecule, showing that the triplet spins are delocalized over the whole pi-conjugated system with an alternate pattern, which is lost in the central region of the polyene chain. The ENDOR investigation strongly supports the hypothesis of localization of the triplet state on one peridinin in each subcluster of the PCP complex, as proposed in [Di Valentin et al. Biochim. Biophys. Acta 1777 (2008) 186-195]. High spin density has been found specifically at the carbon atom at position 12 (see Fig. 1B), which for the peridinin involved in the photo-protective mechanism is in close contact with the water ligand to the chlorophyll a pigment. We suggest that this ligated water molecule, placed at the interface between the chlorophyll-peridinin pair, is functioning as a bridge in the triplet-triplet energy transfer between the two pigments.

FDMR of Carotenoid and Chlorophyll triplets in light-harvesting complex LHCII of spinach

Fluorescence detected magnetic resonance (FDMR) of the light-harvesting complex LHCII of the spinach photosynthetic machinery revealed triplet contributions both from Carotenoids and Chlorophylls. All three carotenoids present in the complex (lutein, neoxanthin and violaxanthin) are evidenced as triplet states in the FDMR signals obtained as variation of the emission intensity of the Chlorophylls in the 680 nm region. The triplets show ⋎D⋎ values of 0.0401, 0.0388 and 0.0382 cm−1. A comparison with the results obtained by ADMR (Absorption Detected Magnetic Resonance) is made and discussed. An interesting concentration effect is discovered and discussed in terms of specific interactions between carotenoids and chlorophyll molecules. Signals are also obtained by microwave sweeping in the Chlorophyll regions and one triplet is detected (⋎D⋎=0.028−0.029 cm−1). The polarization of the carotenoid signals is discussed in terms of singlet-singlet and triplet-triplet energy transfer between carotenoids and chlorophylls, also with the help of double resonance experiments. Double resonance experiments involving both carotenoids and chlorophylls signals gave negative results. It is not possible as a consequence to assess that the chlorophyll whose triplets levels are scanned in the FDMR spectra are functionally connected to the carotenoids.

Cuprizone neurotoxicity, copper deficiency and neurodegeneration

Cuprizone is used to obtain demyelination in mice. Cuprizone-treated mice show symptoms similar to several neurodegenerative disorders such as severe status spongiosus. Although it has a simple chemical formula, its neurotoxic mechanism is still unknown. In this work, we examined both physico-chemical properties and biological effects of cuprizone. Our results indicate that cuprizone has very complicated and misunderstood solution chemistry. Moreover, we show here the inability of cuprizone to cross neither the intestinal epithelial barrier nor the neuronal cell membrane, as well its high tolerability by cultured neurons. If added to mice diet, cuprizone does not accumulate in liver or in brain. Therefore, its neurotoxic effect is explainable only in terms of its capability to chelate copper, leading to chronic copper deficiency.

The [4Fe-4S]-cluster coordination of [FeFe]-hydrogenase maturation protein HydF as revealed by EPR and HYSCORE spectroscopies.

[FeFe] hydrogenases are key enzymes for bio(photo)production of molecular hydrogen, and several efforts are underway to understand how their complex active site is assembled. This site contains a [4Fe-4S]-2Fe cluster and three conserved maturation proteins are required for its biosynthesis. Among them, HydF has a double task of scaffold, in which the dinuclear iron precursor is chemically modified by the two other maturases, and carrier to transfer this unit to a hydrogenase containing a preformed [4Fe-4S]-cluster. This dual role is associated with the capability of HydF to bind and dissociate an iron-sulfur center, due to the presence of the conserved FeS-cluster binding sequence CxHx(46-53)HCxxC. The recently solved three-dimensional structure of HydF from Thermotoga neapolitana described the domain containing the three cysteines which are supposed to bind the FeS cluster, and identified the position of two conserved histidines which could provide the fourth iron ligand. The functional role of two of these cysteines in the activation of [FeFe]-hydrogenases has been confirmed by site-specific mutagenesis. On the other hand, the contribution of the three cysteines to the FeS cluster coordination sphere is still to be demonstrated. Furthermore, the potential role of the two histidines in [FeFe]-hydrogenase maturation has never been addressed, and their involvement as fourth ligand for the cluster coordination is controversial. In this work we combined site-specific mutagenesis with EPR (electron paramagnetic resonance) and HYSCORE (hyperfine sublevel correlation spectroscopy) to assign a role to these conserved residues, in both cluster coordination and hydrogenase maturation/activation, in HydF proteins from different microorganisms.

Carotenoid interactions in peridinin chlorophyll a proteins from dinoflagellates. Evidence for optical excitions and triplet migration

Optical and magnetic resonance spectroscopy has been used to investigate two peridinin–chlorophyll a–protein (PCP) complexes constituting the peripheral antenna of the photosystem of two dinoflagellate species. One protein contains a cluster of four peridinins and one chlorophyll molecule, the other contains two such clusters in a ‘quasi’ dimer of the former. Evidence of intra-cluster excitonic interactions between peridinins is provided by low-temperature absorption and circular dichroism spectra. Peridinin triplets are detected in zero-field magnetic resonance spectroscopy by both optical emission and absorption. Interaction among peridinin molecules causes a fast intra-cluster and a slow inter-cluster triplet migration. The origin of the interactions is discussed by correlating optical and magnetic spectroscopic data.

ODMR of carotenoid and chlorophyll triplets in CP43 and CP47 complexes of spinach

Abstract Optically detected magnetic resonance (ODMR) of the light-harvesting complexes CP43 and CP47 of the spinach photosynthetic machinery revealed triplet states both from carotenoids and chlorophylls. The triplet state of the only carotenoid present in the complexes (β-carotene) is observed by ODMR using fluorescence detection (FDMR) in the main chlorophyll emission band in the 680 nm region, and absorption detection in the visible region. Chlorophyll triplet signals are also obtained by microwave sweeping in the chlorophyll emission and absorption regions. MIA spectra are obtained and discussed for both complexes.