Květoslava Burda

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.

Mössbauer studies of the non-heme iron and cytochrome b559 in a Chlamydomonas reinhardtii PSI- mutant and their interactions with α-tocopherol quinone

Spin and valence states of the non‐heme iron and the heme iron of cytochrome b 559, as well as their interactions with α‐tocopherol quinone (α‐TQ) in photosystem II (PSII) thylakoid membranes prepared from the Chlamydomonas reinhardtii PSI− mutant have been studied using Mössbauer spectroscopy. Both of the iron atoms are in low spin ferrous states. The Debye temperature of the non‐heme is 194 K and of the heme iron is 182 K. The treatment of α‐TQ does not change the spin and the valence states of the non‐heme iron but enhances the covalence of its bonds. α‐TQ oxidizes the heme iron into the high spin Fe3+ state. A possible role of the non‐heme iron and α‐TQ in electron flow through the PSII is discussed.

Molecular dynamics and local electronic states of Sn and Fe in metallocytochrome and metalloporphyrin

In Sn-porphyrin tin appears in the Sn4+ state while in Sn-cytochrome c Sn4+ and Sn2+ states are observed. In Fe-porphyrin iron exhibits temperature-dependent mixed Fe2+-Fe3+ valency. In Fe-cytochrome iron is in the Fe3+ state. The bounded diffusion is observed in Fe-porphyrins above 300 K. The temperature dependence of the metal mean square displacement in cytochrome c and in Sn-porphyrin shows a deviation from the Debye model, which may be explained either by a new vibrational degree of freedom occurring above 150 K or by anharmonicity of the usual vibrations.

Dynamics of electron transfer in photosystem II

Photosystem II, being a constituent of light driven photosynthetic apparatus, is a highly organized pigment-protein-lipid complex. The arrangement of PSII active redox cofactors insures efficiency of electron transfer within it. Donation of electrons extracted from water by the oxygen evolving complex to plastoquinones requires an additional activation energy. In this paper we present theoretical discussion of the anharmonic fluctuations of the protein-lipid matrix of PSII and an experimental evidence showing that the fluctuations are responsible for coupling of its donor and acceptor side. We argue that the fast collective motions liberated at temperatures higher that 200 K are crucial for the two final steps of the water splitting cycle and that one can distinguish three different dynamic regimes of PSII action which are controlled by the timescales of forward electron transfer, which vary with temperature. The three regimes of the dynamical behavior are related to different spatial domains of PSII.

Effect of functionalized and non-functionalized nanodiamond on the morphology and activities of antioxidant enzymes of lung epithelial cells (A549).

The development of nanotechnology opens up new ways for biomedical applications of unmodified and modified diamond nanoparticles which are one of the most popular nanomaterials used in biology, biotechnology, medicine, cosmetics and engineering. They have been applied as diagnostic and therapeutic agents because they can be targeted to and localized in cells causing apoptosis and necrosis. The problem of biocompatibility of nanodiamonds at higher concentrations is thus of primary importance. The first step in the modification of DNPs is usually the introduction of hydrogen groups, which can bind other functional groups. The basic method to introduce -OH groups onto nanoparticles is the Fenton reaction. The aim of this study was to compare the effect of unmodified nanodiamond particles and nanoparticles modified by introduction of -OH groups and etoposide onto their surface reaction on human non-small lung cancer cells. A549 cells were incubated with 2-100μg/ml nanopowders and at 0.6-24μg/ml etoposide in the DMEM medium. We observed a decrease of cells viability and generation of reactive oxygen/ nitrogen species in the cells after incubation, estimated by oxidation of H2DCF-DA and DAF-FM-DA. Modified detonation nanoparticles affected also the cellular content of glutathione and activities of main antioxidant enzymes (glutathione peroxidase, glutathione reductase, glutathione S-transferase, superoxide dismutase and catalase). The results of TEM microscopy show changes in cell morphology. These data demonstrate that modified nanoparticles induce oxidative stress in the target cells.

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.

The influence of very small doses of alpha radiation on the stability of erythrocytes

Our aim was to study the influence of low doses (0.2–4 μGy) of α radiation on the stability of human erythrocytes isolated from healthy and diabetic erythrocytes. Absorption spectroscopy was used to measure the level of red blood cell (RBC) hemolysis, along with Mössbauer spectroscopy, which is a highly specific method suited to monitoring various hemoglobin forms. States of hemoglobin are sensitive to a homeostatic imbalance in red blood cells. Changes in the membrane skeleton organization of irradiated erythrocytes isolated from healthy donors were studied using atomic force microscopy (AFM). Hemolysis, in healthy red blood cells, showed characteristic discontinuities, depending on the α particle flux and the exposure time to the low doses applied. This phenomenon was not observed in severe diabetic cases, which could be a result of modified protein–lipid–sugar complexes and the attenuation/absence of some antioxidative enzymatic processes in their RBC membranes. Similar effects were also observed for red blood cells treated with low doses of neutron and γ‐radiation. AFM measurements demonstrated a reorganization of the RBC membrane skeleton network depending on the time of RBC exposure to α radiation. This suggests that the changes in the activity of the acute defense processes against free radicals which are activated within the erythrocyte membrane irradiated with α‐particles could additionally be up‐ or down regulated by modifications to the membrane–skeleton network. However, even the highest dose of α radiation applied in these studies did not cause any significant changes in the ability of hemoglobin to transport oxygen. Microsc. Res. Tech. 80:131–143, 2017.

Multifractal characterization of morphology of human red blood cells membrane skeleton.

The purpose of this paper is to show applicability of multifractal analysis in investigations of the morphological changes of ultra‐structures of red blood cells (RBCs) membrane skeleton measured using atomic force microscopy (AFM). Human RBCs obtained from healthy and hypertensive donors as well as healthy erythrocytes irradiated with neutrons (45 μGy) were studied. The membrane skeleton of the cells was imaged using AFM in a contact mode. Morphological characterization of the three‐dimensional RBC surfaces was realized by a multifractal method. The nanometre scale study of human RBCs surface morphology revealed a multifractal geometry. The generalized dimensions Dq and the singularity spectrum f(α) provided quantitative values that characterize the local scale properties of their membrane skeleton organization. Surface characterization was made using areal ISO 25178‐2: 2012 topography parameters in combination with AFM topography measurement. The surface structure of human RBCs is complex with hierarchical substructures resulting from the organization of the erythrocyte membrane skeleton. The analysed AFM images confirm a multifractal nature of the surface that could be useful in histology to quantify human RBC architectural changes associated with different disease states. In case of very precise measurements when the red cell surface is not wrinkled even very fine differences can be uncovered as was shown for the erythrocytes treated with a very low dose of ionizing radiation.

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+.