Electron transfer in cyanobacterial photosystem I: I. Physiological and spectroscopic characterization of site-directed mutants in a putative electron transfer pathway from A0 through A1 to FX

The Photosystem I (PS I) reaction center contains two branches of nearly symmetric cofactors bound to the PsaA and PsaB heterodimer. From the x-ray crystal structure it is known that Trp697PsaA and Trp677PsaB are pi-stacked with the head group of the phylloquinones and are H-bonded to Ser692PsaA and Ser672PsaB, whereas Arg694PsaA and Arg674PsaB are involved in a H-bonded network of side groups that connects the binding environments of the phylloquinones and FX. The mutants W697FPsaA, W677FPsaB, S692CPsaA, S672CPsaB, R694APsaA, and R674APsaB were constructed and characterized. All mutants grew photoautotrophically, yet all showed diminished growth rates compared with the wild-type, especially at higher light intensities. EPR and electron nuclear double resonance (ENDOR) studies at both room temperature and in frozen solution showed that the PsaB mutants were virtually identical to the wild-type, whereas significant effects were observed in the PsaA mutants. Spin polarized transient EPR spectra of the P700+A1- radical pair show that none of the mutations causes a significant change in the orientation of the measured phylloquinone. Pulsed ENDOR spectra reveal that the W697FPsaA mutation leads to about a 5% increase in the hyperfine coupling of the methyl group on the phylloquinone ring, whereas the S692CPsaA mutation causes a similar decrease in this coupling. The changes in the methyl hyperfine coupling are also reflected in the transient EPR spectra of P700+A1- and the CW EPR spectra of photoaccumulated A1-. We conclude that: (i) the transient EPR spectra at room temperature are predominantly from radical pairs in the PsaA branch of cofactors; (ii) at low temperature the electron cycle involving P700 and A1 similarly occurs along the PsaA branch of cofactors; and (iii) mutation of amino acids in close contact with the PsaA side quinone leads to changes in the spin density distribution of the reduced quinone observed by EPR.     

Electron transfer in cyanobacterial photosystem I: II. Determination of forward electron transfer rates of site-directed mutants in a putative electron transfer pathway from A0 through A1 to FX

The directionality of electron transfer in Photosystem I (PS I) is investigated using site-directed mutations in the phylloquinone (QK) and FX binding regions of Synnechocystis sp. PCC 6803. The kinetics of forward electron transfer from the secondary acceptor A1 (phylloquinone) were measured in mutants using time-resolved optical difference spectroscopy and transient EPR spectroscopy. In whole cells and PS I complexes of the wild-type both techniques reveal a major, slow kinetic component of tau approximately 300 ns while optical data resolve an additional minor kinetic component of tau approximately 10 ns. Whole cells and PS I complexes from the W697FPsaA and S692CPsaA mutants show a significant slowing of the slow kinetic component, whereas the W677FPsaB and S672CPsaB mutants show a less significant slowing of the fast kinetic component. Transient EPR measurements at 260 K show that the slow phase is approximately 3 times slower than at room temperature. Simulations of the early time behavior of the spin polarization pattern of P700+A1-, in which the decay rate of the pattern is assumed to be negligibly small, reproduce the observed EPR spectra at 260 K during the first 100 ns following laser excitation. Thus any spin polarization from P700+FX- in this time window is very weak. From this it is concluded that the relative amplitude of the fast phase is negligible at 260 K or its rate is much less temperature-dependent than that of the slow component. Together, the results demonstrate that the slow kinetic phase results from electron transfer from QK-A to FX and that this accounts for at least 70% of the electrons. Although the assignment of the fast kinetic phase remains uncertain, it is not strongly temperature dependent and it represents a minor fraction of the electrons being transferred. All of the results point toward asymmetry in electron transfer, and indicate that forward transfer in cyanobacterial PS I is predominantly along the PsaA branch.     

Copper resistance in Desulfovibrio strain R2

A sulfate-reducing bacterium, designated as strain R2, was isolated from wastewater of a ball-bearing manufacturing facility in Tomsk, Western Siberia. This isolate was resistant up to 800 mg Cu/l in the growth medium. By comparison, Cu-resistance of reference cultures of sulfate-reducing bacteria ranged from 50 to 75 mg Cu/l. Growth experiments with strain R2 showed that Cu was an essential trace element and, on one hand, enhanced growth at concentrations up to 10 mg/l but, on the other hand, the growth rate decreased and lag-period extended at copper concentrations of >50 mg/l. Phenotypic characteristics and a 1078 bp nucleotide sequence of the 16S rDNA placed strain R2 within the genus Desulfovibrio. Desulfovibrio R2 carried at least one plasmid of approximately of 23.1 kbp. A 636 bp fragment ot the pcoR gene of the pco operon that encodes Cu resistance was amplified by PCR from plasmid DNA of strain R2. The pco genes are involved in Cu-resistance in some enteric and aerobic soil bacteria. Desulfovibrio R2 is a prospective strain for bioremediation purposes and for developing a homologous system for transformation of Cu-resistance in sulfate-reducing bacteria. This revised version was published online in June 2006 with corrections to the Cover Date.     

Recognition between flexible protein molecules: induced and assisted folding

This review focuses on a very important but little understood type of molecular recognition--the recognition between highly flexible molecular structures. The formation of a specific complex in this case is a dynamic process that can occur through sequential steps of mutual conformational adaptation. This allows modulation of specificity and affinity of interaction in extremely broad ranges. The interacting partners can interact together to form a complex with entirely new properties and produce conformational signal transduction at substantial distance. We show that this type of recognition is frequent in formation of different protein-protein and protein-nucleic acid complexes. It is also characteristic for self-assembly of protein molecules from their unfolded fragments as well as for interaction of molecular chaperones with their substrates and it can be the origin of 'protein misfolding' diseases. Thermodynamic and kinetic features of this type of dynamic recognition and the principles underlying their modeling and analysis are discussed.     

Cerebral blood flow modulates hyperbaric oxygen induced neurotoxicity by neuronal and endothelial nitric oxide

The goal of work was to reveal changes in microcirculation of the rat brain and the role of nitric oxide (NO) in development of seizures at hyperbaric oxygen exposure. The Wistar rats with implanted paired platinum electrodes in left and right striatum were used for experiments. The latency of seizures was defined by the EEG, the cerebral blood flow (CBF) was measured by hydrogen clearance. One group of animals was exposed to a 5-ata oxygen, while the others before oxygen treatment were injected with: Nw-nitro-L-arginine methyl ester (L-NAME), blockator of constitutive NO synthase; 7-nitroindozol (7NI), specific inhibitor of neural NO synthase. The latency of seizures was 41 +/- 1.9 min at 5 ata oxygen exposure. CBF was decreased to 10-14% but before seizures it increased to 23 +/- 9%. L-NAME and 7NI prevented development of hyperoxygen hyperemia and onset of seizures. The results indicate occurrence of hyperbaric oxygen changes of the CBF that modulate neurotoxic effects of NO in neurons as well as in cerebral vessels.     

Institute of Biochemistry and the development of molecular biophyics in the Ukraine

The development of molecular biophysics research in Ukraine is reviewed with historical perspective. Vladimir Belitser and his school were among the pioneers in the studies of protein denaturation and assembly of intermolecular structures. New methods of research of protein conformational transitions and dynamics at equilibrium have been developed at the Palladin Institute of Biochemistry. Presently, the biophysics aspects in different areas of protein research are actively explored. The studies of protein folding in both experimental and theoretical aspects promise especially good prospects.     

Involvement of nitrogen oxide in the cerebral vasoconstriction during respiration with high pressure oxygen

High pressure oxygen evokes a cerebral vasoconstriction and diminishes cerebral blood flow with the aid of mechanisms which are not yet sufficiently studied. We were checking a hypothesis that the hyperbaric oxygen (HBO2) inactivates cerebral nitrogen oxide (NO), interrupts its basal relaxing effect, and evokes a vasoconstriction. In our experiments, HBO2 decreased cerebral blood flow depending on the pressure. Inhibiting the NO-synthase weakened basal vasorelaxation in breathing with atmosphere air and eliminated the vasoconstriction in exposure to the HBO2. Inactivation of O2 prevented the HBO2-induced vasoconstriction. The data obtained reveal that diminishing of cerebral blood flow in HBO is related to the NO inactivation and weakening of its basal vasorelaxing effect. Possible mechanisms of the NO inactivation may involve its reaction with oxygen and superoxide anion which lead to diminishing of the tissue NO concentration and weakening of its vasorelaxing effect.     

Taxonomy and Distribution of Freshwater Pearl Mussels (Unionoida: Margaritiferidae) of the Russian Far East

The freshwater pearl mussel family Margaritiferidae includes 13 extant species, which are all listed by IUCN as endangered or vulnerable taxa. In this study, an extensive spatial sampling of Margaritifera spp. across the Russian Far East (Amur Basin, Kamchatka Peninsula, Kurile Archipelago and Sakhalin Island) was conducted for a revision of their taxonomy and distribution ranges. Based on their DNA sequences, shell and soft tissue morphology, three valid species were identified: Margaritifera dahurica (Middendorff, 1850), M. laevis (Haas, 1910) and M. middendorffi (Rosén, 1926). M. dahurica ranges across the Amur basin and some of the nearest river systems. M. laevis is distributed in Japan, Sakhalin Island and the Kurile Archipelago. M. middendorffi was previously considered an endemic species of the Kamchatka. However, it is widespread in the rivers of Kamchatka, Sakhalin Island, the Kurile Islands (across the Bussol Strait, which is the most significant biogeographical boundary within the archipelago), and, likely, in Japan. The Japanese species M. togakushiensis Kondo & Kobayashi, 2005 seems to be conspecific with M. middendorffi because of similar morphological patterns, small shell size (<100 mm long) and overlapped ranges, but it is in need of a separate revision. Phylogenetic analysis reveals that two NW Pacific margaritiferid species, M. laevis and M. middendorffi, formed a monophyletic 18S rDNA clade together with the North American species M. marrianae and M. falcata. The patterns that were found in these Margaritifera spp. are similar to those of freshwater fishes, indicating multiple colonizations of Eastern Asia by different mitochondrial lineages, including an ancient Beringian exchange between freshwater faunas across the Pacific.     

Microsporidian Parasites Found in the Hemolymph of Four Baikalian Endemic Amphipods

At present, approximately 187 genera and over 1300 species of Microsporidia have been described, among which almost half infect aquatic species and approximately 50 genera potentially infect aquatic arthropods. Lake Baikal is the deepest and one of the oldest lakes in the world, and it has a rich endemic fauna with a predominance of arthropods. Among the arthropods living in this lake, amphipods (Crustacea) are the most dominant group and are represented by more than 350 endemic species. Baikalian amphipods inhabit almost all depths and all types of substrates. The age and geographical isolation of this group creates excellent opportunities for studying the diversity, evolution and genetics of host-parasite relationships. However, despite more than 150 years of study, data investigating the microsporidia of Lake Baikal remain incomplete. In this study, we used molecular genetic analyses to detect microsporidia in the hemolymph of several endemic species of amphipods from Lake Baikal. We provide the first evidence that microsporidian species belonging to three genera (Microsporidium, Dictyocoela and Nosema) are present in the hemolymph of Baikalian endemic amphipods. In the hemolymph of Eulimnogammarus verrucosus, we detected SSU rDNA of microsporidia belonging to the genus Nozema. In the hemolymph of Pallasea cancellous, we found the DNA of Microsporidium sp. similar to that in other Baikalian endemic amphipods; Dictyocoela sp. was found in the hemolymph of Eulimnogammarus marituji and Acanthogammarus lappaceus longispinus.