Joanna Fiedor

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.

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

Abstract 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 174-methyl-132-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. β-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 lifetimes of the pigment or efficient intersystem crossing (or both).

Photodynamics of the Bacteriochlorophyll–Carotenoid System. 1. Bacteriochlorophyll-photosensitized Oxygenation of β-Carotene in Acetone¶

Carotenoids are well‐known physical quenchers of chlorophyll excited states and reactive oxygen species both in vivo and in vitro. They may also be involved in chemical quenching undergoing, e.g. isomerizations or oxidations. We have found that β‐carotene (Car) in aerobic acetone is rapidly oxygenated under strong illumination with red light (λexc≥ 630 nm) in the presence of bacteriopheophytin a. At the same time the photosensitizer undergoes only slight (<10%) photodegradation. By preparative high‐performance liquid chromatography as many as seven major products of oxygen attachment to Car have been isolated. Their molecular masses show that Car sequentially accumulates up to six oxygen atoms while its C40‐skeleton remains intact.

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-β-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.

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.

The dynamics of the non-heme iron in bacterial reaction centers from Rhodobacter sphaeroides.

We investigate the dynamical properties of the non-heme iron (NHFe) in His-tagged photosynthetic bacterial reaction centers (RCs) isolated from Rhodobacter (Rb.) sphaeroides. Mössbauer spectroscopy and nuclear inelastic scattering of synchrotron radiation (NIS) were applied to monitor the arrangement and flexibility of the NHFe binding site. In His-tagged RCs, NHFe was stabilized only in a high spin ferrous state. Its hyperfine parameters (IS=1.06±0.01mm/s and QS=2.12±0.01mm/s), and Debye temperature (θ(D0)~167K) are comparable to those detected for the high spin state of NHFe in non-His-tagged RCs. For the first time, pure vibrational modes characteristic of NHFe in a high spin ferrous state are revealed. The vibrational density of states (DOS) shows some maxima between 22 and 33meV, 33 and 42meV, and 53 and 60meV and a very sharp one at 44.5meV. In addition, we observe a large contribution of vibrational modes at low energies. This iron atom is directly connected to the protein matrix via all its ligands, and it is therefore extremely sensitive to the collective motions of the RC protein core. A comparison of the DOS spectra of His-tagged and non-His-tagged RCs from Rb. sphaeroides shows that in the latter case the spectrum was overlapped by the vibrations of the heme iron of residual cytochrome c(2), and a low spin state of NHFe in addition to its high spin one. This enabled us to pin-point vibrations characteristic for the low spin state of NHFe.

Coupling of collective motions of the protein matrix to vibrations of the non-heme iron in bacterial photosynthetic reaction centers

Non-heme iron is a conservative component of type II photosynthetic reaction centers of unknown function. We found that in the reaction center from Rba. sphaeroides it exists in two forms, high and low spin ferrous states, whereas in Rsp. rubrum mostly in a low spin state, in line with our earlier finding of its low spin state in the algal photosystem II reaction center (Burda et al., 2003). The temperature dependence of the non-heme iron displacement studied by Mössbauer spectroscopy shows that the surrounding of the high spin iron is more flexible (Debye temperature ~165K) than that of the low spin atom (~207K). Nuclear inelastic scattering measurements of the collective motions in the Rba. sphaeroides reaction center show that the density of vibrational states, originating from non-heme iron, has well-separated modes between lower (4-17meV) and higher (17-25meV) energies while in the one from Rsp. rubrum its distribution is more uniform with only little contribution of low energy (~6meV) vibrations. It is the first experimental evidence that the fluctuations of the protein matrix in type II reaction center are correlated to the spin state of non-heme iron. We propose a simple mechanism in which the spin state of non-heme iron directly determines the strength of coupling between the two quinone acceptors (Q(A) and Q(B)) and fast collective motions of protein matrix that play a crucial role in activation and regulation of the electron and proton transfer between these two quinones. We suggest that hydrogen bond network on the acceptor side of reaction center is responsible for stabilization of non-heme iron in different spin states.

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.

Influence of Cd2+ on the spin state of non-heme iron and on protein local motions in reactions centers from purple photosynthetic bacterium Rhodospirilium rubrum

Non-heme Fe is a conservative component of the Q-type photosynthetic reaction centers but its function remains unknown. Applying Mossbauer spectroscopy we show that in Rhodospirillum rubrum the non-heme Fe exists mostly in a ferrous low spin state. The binding of Cd2+ ions in the vicinity of the quinone-Fe complex changes the high spin state of the non-heme Fe into a low spin one characterized by hyperfine parameters similar to those obtained for the non-heme Fe low spin state in untreated reaction centers, as confirmed by Mossbauer measurements. The nuclear inelastic scattering of synchrotron radiation experiments show that the contribution of vibrations at low energies, between 3-15 meV, activated at 240 K are damped in the bacterial reaction centers treated with CdCl2. No influence of Cd2+ ions is observed on the soft vibrational states at 60 K. These results suggest that binding of cadmium cations within the reaction centers may enhance decoupling of the non-heme Fe from the surrounding protein matrix at temperatures higher than 200 K, what can explain the slowing down of electron transfer between the QA and QB quinones by Cd2+.

Quantification of purple non-sulphur phototrophic bacteria and their photosynthetic structures by means of total reflection X-ray fluorescence spectrometry (TXRF)

Phototrophic bacteria are metabolically diverse microorganisms that attract substantial attention due to their growing potential in scientific and industrial applications. In the present study, total reflection X-ray fluorescence (TXRF) spectrometry was applied to perform a comprehensive analysis of the trace elements content of purple non-sulphur phototrophic bacteria, their chromatophores and selected photosynthetic structures in response to alterations in oxygen growth conditions. There is a lack of consistent information on the content of microelements, their distribution and in particular correlations between them. This analysis, carried out on the Rhodobacter sphaeroides species aims to rectify this. Qualitative examination revealed the presence of microelements generally not considered as basic in the bacterial ionome. Quantitative inspection pointed to Fe as the major trace element in this phototrophic species irrespective of growth conditions (sample type). The K/Rb and Ca/Sr ratios were determined for the first time for bacteria and their photosynthetic membranes. Finally, the ionomic approach to elemental accumulation followed by statistical analysis revealed intriguing relationships between the elements within cells and phototrophic membranes. The vast potential and usefulness of the TXRF technique in a wide range of biological and environmental applications is underlined.