New Facts about CdCl2 Action on the Photosynthetic Apparatus of Spinach Chloroplasts and Its Comparison with HgCl2 Action

Using EPR spectroscopy it was found that CdCl₂ and HgCl₂ interact (1) with the intermediates , i.e. with the tyrosine radicals on the donor side of photosystem (PS) 2 situated in the 161^st position in D₁ and D₂ proteins; (2) with the primary donor of PS1 (P700) whereby the oxidation of chlorophyll (Chl) a dimer in the reaction centre of PS1 occurs yet in the dark; (3) with the manganese cluster which is situated in the oxygen evolving complex. Due to these interactions of investigated metal chlorides with the photosynthetic apparatus, the interruption of the photosynthetic electron transport through photosynthetic centres occurs. Monitoring of time dependence of EPR signal I of chloroplasts treated with CdCl₂ or HgCl₂ after switching off the light suggests that all mechanisms, i.e. direct, cyclic, and non-cyclic reductions of P700⁺ are damaged. The formation of complexes between mercury or cadmium ions and amino acid residues constituting photosynthetic peptides was suggested as possible mechanism of their inhibitory action. The higher HgCl₂ efficiency in comparison with that of CdCl₂ was explained by higher ability of mercury ions to form complexes with amino acids, what was demonstrated by their apparent binding constants: K = 10 200 M^–1 for Hg²⁺ ions, and K = 3 700 M^–1 for Cd²⁺ ions.     

In vivo interaction of trivalent chromium with yeast plasma membrane, as revealed by EPR spectroscopy

The in vivo effects of CrCl(3) on an ergosterol-producing 33 erg(+) strain of the eukaryotic yeast Candida albicans, and on its ergosterol-deficient erg(-)2 mutant, were studied by using electron paramagnetic resonance spectroscopy. A 5-doxylstearic acid spin probe was applied to label the membranes. The absence of ergosterol, an increased accumulation of Delta(8) sterols, a decreased fatty acid chainlength and a lower proportion of unsaturated fatty acids of the erg(-)2 mutant resulted in a higher membrane rigidity and an increased sensitivity to Cr(III) than those of the parental 33 erg(+) strain. Cr(III) significantly increased the fluidity of the spin labelled membranes, this being more pronounced for the erg(-)2 mutant. The break in the slopes measured for the erg(-)2 mutant was decreased (DeltaAT approximately 4 degrees C) from 17 to 13 degrees C. Cr(III) treatment for 10 h caused a loss of metabolites adsorbing at 260 nm: this loss was 40% for 33 erg(+) and 60% for erg(-)2. This decriptification process might be the main cause of growth inhibition and cell killing by the impermeable Cr(III) ions.     

Diphenylene iodonium sensitivity of a solubilized membrane enzyme from rose cells

Summary An NADH-specific oxidation reduction enzyme has been partially purified from rose cell microsomes by aqueous two-phase partitioning, ultracentrifugation, and ion-exchange chromatography, on the basis of the enzyme's ability to activate lucigenin chemiluminescence in the presence of NADH. The enzyme showed strong similarity to a plasma membrane NADH oxidase (superoxide synthase as assayed by lucigenin chemiluminescence; T. M. Murphy and C.-K. Auh, Plant Physiol. 110: 621–629, 1996) in its response to substrate, to Triton X-100, and to diphenylene iodonium, an inhibitor of mammalian neutrophil NADPH oxidase and other flavoenzymes. However, its fluorescence spectrum was not characteristic of flavins and instead was similar to that of pterins. Thus inhibition of an enzyme-catalyzed reaction by diphenylene iodonium does not necessarily imply that the reaction is catalyzed by NADPH oxidase or another flavoenzyme. Superoxide synthesis catalyzed by the enzyme preparation was very low but could be increased at least twofold by the addition of a quinone, menadione. This suggests the enzyme acting in conjunction with a natural quinone could produce activated oxygen species in stressed plant cells.Abbreviation DPI diphenylene iodonium     

Spectroscopic and magnetic properties of a Ni(II) complex with citric acid

The complex compound K₂[Ni(cit)(H₂O)₂]₂·4H₂O (cit = triionized citrate ion) seems to be a good model for the investigation of Ni(II)/citrate interactions that are of interest in relation to nickel metabolism and bioaccumulation. Its infrared and Raman spectra were recorded and analyzed on the basis of its structural peculiarities. The magnetic susceptibility, investigated in the temperature range between 1.9 and 300 K, shows the absence of magnetic interactions between the two metal centers present in this structure.     

Characterization of carotenes in a combination of a C18 HPLC column with isocratic elution and absorption spectra with a photodiode-array detector

Carotenes have attracted much attention in recent years for their biological function in processes such as photosynthesis. The characterization of carotenes is difficult, however, because they consist of only carbon and hydrogen atoms, without oxygen. In the present study, we systematically examined the chemical structures of more than 30 carotenes, including most of the carotenes found in phototrophic organisms, and observed their elution order using a Novapak C₁₈ HPLC column with simple isocratic elution. The elution order of the carotenes was C₃₀, C₄₀,C₄₅ then C₅₀. The C₄₀ carotenes with fewer conjugated double bonds (N) had longer retention times. With respect to the end groups, the carotenes eluted in the following order: φ, Ψ, ∈ then β end groups. Furthermore, absorption spectra in the HPLC eluent used were recorded with a photodiode-array detector. A greater N value was associated with a longer absorption maximum wavelength. Since the conjugated end groups (φ and β) influenced the absorption spectra and the non-conjugated end groups (Ψ and ∈) did not, the number of conjugated end groups (zero, one and two) was clearly distinguishable. Therefore, the chemical structures of carotenes can be easily determined by a combination of the HPLC retention times and the absorption spectra. This revised version was published online in June 2006 with corrections to the Cover Date.     

Di-cluster, seven-iron ferredoxins from hyperthermophilic Sulfolobales

 Seven-iron ferredoxins from the thermoacidophilic archaea Acidianus ambivalens, A. infernus, Metalosphaera prunae and Sulfolobus metallicus were extensively characterised, allowing study of their expression under aerobic and anaerobic growth conditions as well as the putative role in thermal stability of a recently described zinc centre. The archaeon S. metallicus was found to express, under the same growth conditions, two ferredoxins in almost identical amounts, a novelty among Archaea. Most interestingly, these two ferredoxins differ at the N-terminal amino acid sequence in that one has a zinc binding motif (FdA) and the other does not (FdB); in agreement with these findings, FdA contains a zinc ion and FdB does not. These two ferredoxins have identical thermal stabilities, indicating that the zinc atom is not determinant in the protein thermostability. Further, the presence of the additional zinc centre does not interfere with the redox properties of the iron-sulfur clusters since their reduction potentials are almost identical. From the other three archaea, independently of the growth mode in respect to oxygen, only a single zinc-containing ferredoxin was found. EPR studies on the purified proteins, both in the oxidised and dithionite reduced states, allowed the identification of one [3Fe-4S]^1+/0 centre and one [4Fe-4S]^2+/1+ centre in all proteins studied. The complete sequence of A. ambivalens ferredoxin is reported. Together with the data gathered in this study, the properties of the seven-iron ferredoxins from Sulfolobales so far known are re-discussed. Received: 10 June 1998 / Accepted: 25 June 1998     

离子注入不同辐射敏感性微生物自由基与存活关系的研究

以不同辐射敏感性微生物辐射异常微球菌和大肠杆菌为试材,用电子自旋共振波谱法研究了离子注入后在两种微生物细胞内产生的自由基及其存活的关系。     

Inhibition of Membrane Lipid Peroxidation by a Radical Scavenging Mechanism: a Novel Function for Hydroxyl-Containing Ionophores

In the present study we show that K⁺/H⁺ hydroxyl-containing ionophores lasalocid-A (LAS) and nigericin (NIG) in the nanomolar concentration range, inhibit Fe²⁺-citrate and 2,2'-azobis(2-amidinopropane) di-hydrochloride (ABAP)-induced lipid peroxidation in intact rat liver mitochondria and in egg phosphatidyl-choline (PC) liposomes containing negatively charged lipids—dicetyl phosphate (DCP) or cardiolipin (CL)—and KCl as the osmotic support. In addition, monensin (MON), a hydroxyl-containing ionophore with higher affinity for Na⁺ than for K⁺, promotes a similar effect when NaCl is the osmotic support. The protective effect of the ionophores is not observed when the osmolyte is sucrose. Lipid peroxidation was evidenced by mitochondrial swelling, antimycin A-insensitive O₂ consumption, formation of thiobarbituric acid-reactive substances (TBARS), conjugated dienes, and electron paramagnetic resonance (EPR) spectra of an incorporated lipid spin probe. A time-dependent decay of spin label EPR signal is observed as a consequence of lipid peroxidation induced by both inductor systems in liposomes. Nitroxide destruction is inhibited by buty-lated hydroxytoluene, a known antioxidant, and by the hydroxyl-containing ionophores. In contrast, vali-nomycin (VAL), which does not possess alcoholic groups, does not display this protective effect. Effective order parameters (S_eff), determined from the spectra of an incorporated spin label are larger in the presence of salt and display a small increase upon addition of the ionophores, as a result of the increase of counter ion concentration at the negatively charged bilayer surface. This condition leads to increased formation of the ion-ionophore complex, the membrane binding (uncharged) species. The membrane-incorporated complex is the active species in the lipid peroxidation inhibiting process. Studies in aqueous solution (in the absence of membranes) showed that NIG and LAS, but not VAL, decrease the Fe²⁺-citrate-induced production of radicals derived from piperazine-based buffers, demonstrating their property as radical scavengers. Both Fe²⁺-citrate and ABAP promote a much more pronounced decrease of LAS fluorescence in PC/CL liposomes than in dimyristoyl phosphatidyl-choline (DMPC, saturated phospholipid)-DCP liposomes, indicating that the ionophore also scavenges lipid peroxyl radicals. A slow decrease of fluorescence is observed in the latter system, for all lipid compositions in sucrose medium, and in the absence of membranes, indicating that the primary radicals stemming from both inductors also attack the ionophore. Altogether, the data lead to the conclusion that the membrane-incorporated cation complexes of NIG, LAS and MON inhibit lipid peroxidation by blocking initiation and propagation reactions in the lipid phase via a free radical scavenging mechanism, very likely due to the presence of alcoholic hydroxyl groups in all three molecules and to the attack of the aromatic moiety of LAS.     

Simultaneous detection of spin-coupled and decoupled QA– EPR-signals in Photosystem II complexes isolated with isoelectric focusing

The Photosystem II multisubunit protein complex can be extracted from thylakoid membranes with non-ionic detergents and subjected to various spectroscopical and biochemical investigations. This paper shows that after extraction with dodecyl--D-maltoside, several Photosystem II complexes could be resolved by isoelectric focusing. Structurally, the various Photosystem II complexes differed from each other in polypeptide composition, especially with regard to the chlorophyll a/b-binding proteins, which gave rise to differing isoelectric points. Functionally, the various Photosystem II complexes differed from each other on the acceptor side, as judged by acceptor side-dependent electron transfer and electron paramagnetic resonance (EPR). The Q_A ^- Fe2+-signal (g = 1.84), arising from Q_A ^- spin-coupled to the acceptor-side iron, and a radical signal arising from decoupled Q_A ^- (g = 2.0045) could be detected simultaneously in some of the Photosystem II complexes, and the amount of each of the two signals were inversely related. The results are discussed in relation to previously known heterogeneities in Photosystem II.     

Studies on the antioxidant activity of some chromonylrhodanine derivatives

Fifteen chromonylrhodamine derivatives (CRs) were synthesized and the antioxidant activity levels were evaluated for the first time. The antioxidant activity potencies of these chromone derivatives were evaluated towards superoxide anion radicals, hydroxyl radicals and 2,2‐diphenyl‐1‐picrylhydrazyl radicals. Also, the total antioxidant capacity of the tested compounds was measured using the ferric‐ferrozine assay. The antioxidant activities were investigated using a chemiluminescence (CL) assay, spectrophotometry measurements, direct electron paramagnetic resonance (EPR) and the EPR spin‐trapping technique. The 5,5‐dimethyl‐ 1‐pyrroline‐1‐oxide (DMPO) was applied as spin trap. Eleven of the 15 chromone compounds exhibited a decrease in the CL accompanying the superoxide anion radical produced in anhydrous dimethylsulfoxide (DMSO), ranging from 71–94% at concentration of 1 mmol /L; four of these compounds enhanced light emission in the range 231–672%. Similarly, these compounds caused 28–58% inhibition in the intensity of the DMPO‐OOH radical EPR signal and the DMPO‐OH radical (from 12–48%). Furthermore, three of these compounds showed very good antioxidant response towards the DPPH radical (EC₅₀: 0.51–0.56 µmol/L) and the high reduction potentials. These findings demonstrate that the chromone compounds tested may be considered as effective free radicals scavengers, a finding that is of great pharmacological importance. Copyright © 2014 John Wiley & Sons, Ltd.