Triplet-driven chemical reactivity of ß-carotene and its biological implications.

The endoperoxides of ß-carotene (ßCar-EPOs) are regarded as main products of the chemical deactivation of 1O2 by ß-carotene, one of the most important antioxidants, following a concerted singlet-singlet reaction. Here we challenge this view by showing that ßCar-EPOs are formed in the absence of 1O2 in a non-concerted triplet-triplet reaction: 3O2 + 3ß-carotene → ßCar-EPOs, in which 3ß-carotene manifests a strong biradical character. Thus, the reactivity of ß-carotene towards oxygen is governed by its excited triplet state. ßCar-EPOs, while being stable in the dark, are photochemically labile, and are a rare example of nonaromatic endoperoxides that release 1O2, again not in a concerted reaction. Their light-induced breakdown triggers an avalanche of free radicals, which accounts for the pro-oxidant activity of ß-carotene and the puzzling swap from its anti- to pro-oxidant features. Furthermore, we show that ßCar-EPOs, and carotenoids in general, weakly sensitize 1O2. These findings underlie the key role of the triplet state in determining the chemical and photophysical features of ß-carotene. They shake up the prevailing models of carotenoid photophysics, the anti-oxidant functioning of ß-carotene, and the role of 1O2 in chemical signaling in biological photosynthetic systems. ßCar-EPOs and their degradation products are not markers of 1O2 and oxidative stress but of the overproduction of extremely hazardous chlorophyll triplets in photosystems. Hence, the chemical signaling of overexcitation of the photosynthetic apparatus is based on a 3chlorophyll-3ß-carotene relay, rather than on extremely short-lived 1O2.

ß-Carotene-Induced Alterations in Haemoglobin Affinity to O2.

ß-Carotene (ß-Crt) can be dispersed in hydrophobic regions of the membrane of red blood cells (RBC). Its location, orientation and distribution strongly depend on carotenoid concentration. In the present pilot trial (six human subjects involved), it is demonstrated that incubation of RBCs with ß-Crt (1.8 × 107 ß-Crt molecules per RBC, 50 µmol/L) results in expansion of the membrane of RBCs and slight elongation of the cell. The changes are of statistical significance, as verified by the Wilcoxon test at p < 0.05. They indicate (i) a highly random orientation and location of ß-Crt inside the membrane and (ii) a tendency for its interaction with membrane skeleton proteins. The accompanying effect of decreased RBC resistance to lysis is possibly a result of the incorrect functioning of ion channels due to their modification/disruption. At higher ß-Crt concentrations, its clustering inside membranes may occur, leading to further alterations in the shape and size of RBCs, with the most pronounced changes observed at 1.8 × 108 ß-Crt molecules per RBC (500 µmol/L). Due to the reduced permeability of ions, such membranes exhibit increased resistance to haemolysis. Finally, we show that interactions of ß-Crt with the membrane of RBCs lead to an alteration in haemoglobin-oxygen affinity, shifting the oxyhaemoglobin dissociation curve toward higher oxygen partial pressures. If the impact of ß-Crt on a curve course is confirmed in vivo, one may consider its role in the fine tuning of O2 transportation to tissues. Hence, at low concentrations, providing unchanged elastic and functional properties of RBCs, it could serve as a beneficial agent in optimising heart performance and cardiovascular load.

Seasonal contribution of assessed sources to submicron and fine particulate matter in a Central European urban area.

This study presents the air pollution findings of the submicron (PM1) and fine (PM2.5) particulate matter. The submicron particles are entirely absorbed by the human body and they cause the greatest health risk. For the PM2.5 concentration, there are yearly and/or daily limit values regulations by the European Union (EU) and World Health Organization (WHO). There are no such regulations for PM1 but for health risk reason the knowledge of its concentration is important. This paper presents the seasonal concentration contribution of PM1 and PM2.5, their chemical composition and assessed three basic sources. Daily samples of both fractions were collected from 2nd July 2016 to 27th February 2017 in Krakow, Poland. Apart from PM1 and PM2.5 the concentration of 16 elements, 8 ions and BC for each samples were measured. Based on these chemical species the positive matrix factorization (PMF) receptor modeling was used for the determination of three main sources contribution to the PM1 and PM2.5 concentrations. Daily average concentrations of PM2.5 were 12 µg/m3 in summer and 60 µg/m3 in winter. For PM1 it was 6.9 µg/m3 in summer and 17.3 µg/m3 in winter. These data show a significant difference in percentage contribution of PM1 in PM2.5 in summer (58%) and in winter (29%). For the combustion source, the concentrations calculated from PMF modeling in winter were 4.8 µg/m3 for PM1 and 31 µg/m3 for PM2.5. In summer, the concentrations were smaller than 1 µg/m3 for both fractions. Secondary aerosols' concentration for PM1 was 3.4 µg/m3 in summer and 11 µg/m3 in winter - for PM2.5 these were 7.1 µg/m3 and 17 µg/m3 respectively. The third source - soil, industry and traffic together, had small seasonal variation: for PM1 it was from 1.4 to 1.8 µg/m3 and for PM2.5 from 4.7 to 7.9 µg/m3.

Chemical content and estimated sources of fine fraction of particulate matter collected in Krakow.

The monitored level of pollution remains high in Krakow, Poland. Alerts regarding increased levels of pollution, which advise asthmatics, the elderly, and children to limit their exposure to open air, continue to be issued on numerous days. In this work, seasonal variations in PM2.5 (particulate matter containing particles with aerodynamic diameter no higher than 2.5 µm) concentrations are shown. An increasing trend is reported, which is enhanced during the colder seasons. The mean PM2.5 concentrations in Krakow exceeded the target value of 25 µg/m3 specified for 2015 in the spring, autumn, and winter seasons. For this reason, particulate matter pollution is of special concern. Elemental concentrations as well as the presence of black carbon (BC) and black smoke (BS) in PM2.5 samples were determined. Seasonal variations of Cl, K, Ca, Ti, Mn, Fe, Cu, Zn, Br, Rb, Sr, and Pb concentrations were observed whereas V, Cr, Ni, BC, and BS concentrations did not significantly change with the time of year. Seven factors were identified by the positive matrix factorization (PMF) technique, and one was non-identified. They were attributed to the following sources of pollution: steel industry, traffic (diesel exhaust), traffic (gasoline exhaust, brake wear), road dust, construction dust, combustion (biomass, coal), and non-ferrous metallurgical industry. The last, non-identified source, could be attributed to secondary aerosols. It is worth to mention that combustion shows significant seasonal variations with a high impact in winter. The reported results of the completed studies may significantly aid in solving air quality issues in the city by highlighting major sources of air pollution.

Effects of Molecular Symmetry on the Electronic Transitions in Carotenoids.

The aim of this work is the verification of symmetry effects on the electronic absorption spectra of carotenoids. The symmetry breaking in cis-ß-carotenes and in carotenoids with nonlinear π-electron system is of virtually no effect on the dark transitions in these pigments, in spite of the loss of the inversion center and evident changes in their electronic structure. In the cis isomers, the S2 state couples with the higher excited states and the extent of this coupling depends on the position of the cis bend. A confrontation of symmetry properties of carotenoids with their electronic absorption and IR and Raman spectra shows that they belong to the C1 or C2 but not the C2h symmetry group, as commonly assumed. In these realistic symmetries all the electronic transitions are symmetry-allowed and the absence of some transitions, such as the dark S0 → S1 transition, must have another physical origin. Most likely it is a severe deformation of the carotenoid molecule in the S1 state, unachievable directly from the ground state, which means that the Franck-Condon factors for a vertical S0 → S1 transition are negligible because the final state is massively displaced along the vibrational coordinates. The implications of our findings have an impact on the understanding of the photophysics and functioning of carotenoids.

Potential role of carotenoids as antioxidants in human health and disease.

Carotenoids constitute a ubiquitous group of isoprenoid pigments. They are very efficient physical quenchers of singlet oxygen and scavengers of other reactive oxygen species. Carotenoids can also act as chemical quenchers undergoing irreversible oxygenation. The molecular mechanisms underlying these reactions are still not fully understood, especially in the context of the anti- and pro-oxidant activity of carotenoids, which, although not synthesized by humans and animals, are also present in their blood and tissues, contributing to a number of biochemical processes. The antioxidant potential of carotenoids is of particular significance to human health, due to the fact that losing antioxidant-reactive oxygen species balance results in "oxidative stress", a critical factor of the pathogenic processes of various chronic disorders. Data coming from epidemiological studies and clinical trials strongly support the observation that adequate carotenoid supplementation may significantly reduce the risk of several disorders mediated by reactive oxygen species. Here, we would like to highlight the beneficial (protective) effects of dietary carotenoid intake in exemplary widespread modern civilization diseases, i.e., cancer, cardiovascular or photosensitivity disorders, in the context of carotenoids' unique antioxidative properties.

Excitation energy trapping and dissipation by Ni-substituted bacteriochlorophyll a in reconstituted LH1 complexes from Rhodospirillum rubrum.

Bacteriochlorophyll a with Ni(2+) replacing the central Mg(2+) ion was used as an ultrafast excitation energy dissipation center in reconstituted bacterial LH1 complexes. B870, a carotenoid-less LH1 complex, and B880, an LH1 complex containing spheroidene, were obtained via reconstitution from the subunits isolated from chromatophores of Rhodospirillum rubrum . Ni-substituted bacteriochlorophyll a added to the reconstitution mixture partially substituted the native pigment in both forms of LH1. The excited-state dynamics of the reconstituted LH1 complexes were probed by femtosecond pump-probe transient absorption spectroscopy in the visible and near-infrared spectral region. Spheroidene-binding B880 containing no excitation dissipation centers displayed complex dynamics in the time range of 0.1-10 ps, reflecting internal conversion and intersystem crossing in the carotenoid, exciton relaxation in BChl complement, and energy transfer from carotenoid to the latter. In B870, some aggregation-induced excitation energy quenching was present. The binding of Ni-BChl a to both B870 and B880 resulted in strong quenching of the excited states with main deexcitation lifetime of ca. 2 ps. The LH1 excited-state lifetime could be modeled with an intrinsic decay time constant in Ni-substituted bacteriochlorophyll a of 160 fs. The presence of carotenoid in LH1 did not influence the kinetics of energy trapping by Ni-BChl unless the carotenoid was directly excited, in which case the kinetics was limited by a slower carotenoid S1 to bacteriochlorophyll energy transfer.

Antioxidant effects of carotenoids in a model pigment-protein complex.

The effect of carotenoids on stability of model photosynthetic pigment-protein complexes subjected to chemical oxidation with hydrogen peroxide or potassium ferricyanide was investigated. The oxidation of carotenoid-less and carotenoid-containing complexes was conducted in the presence or absence of ascorbic acid. The progress of the reactions was monitored by use of absorption and fluorescence spectroscopy. Our results show that carotenoids may significantly enhance the stability of photosynthetic complexes against oxidation and their protective (antioxidant) effect depends on the type of the oxidant.

Tuning the thermodynamics of association of transmembrane helices.

Modular photosynthetic LH1 complex is applied as a model system to investigate the thermodynamics of a self-assembling membrane protein and the effects of cosolvents and cofactor (carotenoid) on the process. Native chromophores of LH1, bacteriochlorophyll, and carotenoid are excellent intrinsic spectroscopic reporter molecules. Their presence allows us to follow the association of transmembrane helices of LH1, without the use of any external markers, by electronic absorption/emission and circular dichroism. Furthermore, the assembly correctness can be monitored by the intracomplex energy transfer. Both the cosolvent and carotenoid markedly affect DeltaH degrees and DeltaS degrees associated with the complex formation in detergent, but the driving force of the process remains almost constant due to an efficient enthalpy-entropy compensation in the system. In the absence of cosolvent and cofactor, the energy of interactions between transmembrane helices in LH1 equals -580 kJ/mol. DeltaH degrees drastically increases upon the addition of acetone (-1160 kJ/mol) and carotenoid (-1900 kJ/mol), whereas DeltaS degrees lowers from +1.5 kJ/mol.K to -0.4 kJ/mol.K and to -2.6 kJ/mol.K, respectively. The stabilization of the ensemble by cofactor seems to be due to the pi-pi stacking of aromatic residues of LH1 polypeptides with the carotenoid pi-electron system. The cosolvent, lowering the medium permittivity and thus enhancing helix-helix interactions, has an ordering effect on the system (DeltaS degrees<0). This effect of cosolvent on DeltaH degrees and DeltaS degrees of association of transmembrane helices is relevant for crystallization of membrane proteins, as it explains in thermodynamic terms the action of amphiphiles used for crystallization of membrane proteins in the micellar phase.

Cyclic endoperoxides of beta-carotene, potential pro-oxidants, as products of chemical quenching of singlet oxygen.

Photoprotection by carotenoids is generally considered to be based on the photophysical quenching of triplets and singlet oxygen. There is also accumulating evidence of an alternative, chemical quenching of triplets and singlet oxygen by carotenoids. We report the identification of relatively stable cyclic mono- and diendoperoxides as first products of such an alternative reaction. Nevertheless, these species remain reactive and in the dark cause autooxidation of beta-carotene in our model system. Their formation could explain the intriguing pro-oxidant and cytotoxic activity of carotenoids.

Photodynamics of the bacteriochlorophyll-carotenoid system. 2. Influence of central metal, solvent and beta-carotene on photobleaching of bacteriochlorophyll derivatives.

Bacteriochlorophyll (BChl) derivatives (with central Mg replaced by metal "M") ([M]-BChl with M = 2H, Mg, Zn, Pd, Cu) have been investigated for their photodynamic capacity and stability toward photodegradation in organic solvents and aqueous micellar solution. A protocol has been developed for screening new sensitizers. BChl and [Zn]-BChl are efficient sensitizers, but they are also quickly degraded by the reactive oxygen species (ROS) produced by autosensitization, as well as by hetero-sensitization with 17(4)-methyl-13(2)-demethoxycarbonyl-pheophorbide a (MPP). Photostable [Cu]-BChl is a poor sensitizer, whereas [Pd]-BChl and bacteriopheophytin a are not only very efficient sensitizers but are also very stable toward ROS. beta-Carotene is no efficient physical quencher of ROS in the system; rather, it acts as a photochemical quencher that competes with [M]-BChl and undergoes photooxygenation at high rates. Photolability seems to depend on the pigment oxidation potential and, in parallel, on the presence of central metals preferring coordination numbers higher than 4, whereas photodynamic capacity depends on long excited state life-times of the pigment or efficient intersystem crossing (or both).