Slow electrogenic events in the cytochrome bc1-complex of Rhodobacter sphaeroides. The electron transfer between cytochrome b hemes can be non-electrogenic.

The flash-induced formation of transmembrane electric potential differences (measured by carotenoid bandshift) and redox changes of cytochrome bh (b561) were monitored spectrophotometrically in Rb. sphaeroides chromatophores in a pH range from 7.5 to 10.0. It is shown that in the presence of antimycin A and at pH less than 8.3 the myxothiazol-sensitive, antimycin-insensitive component of the carotenoid bandshift is kinetically coupled to cytochrome bh reduction. The kinetics of both processes can be described by a single exponent with a rise time of about 10 ms. Alkalization of the medium (8.3 less than or equal to pH less than or equal to 9.2) causes the appearance of an additional constituent in this phase of the carotenoid response with the rise time varying in the range of 100-300 ms. With a further pH increase (pH greater than 9.2), the electrogenic constituent, kinetically linked to cytochrome bh reduction, diminishes. The obtained data are discussed within the framework of the scheme, assuming that the electron transfer between bl and bh hemes in the bc1 complex is, under certain conditions, accompanied by proton transfer in the same direction.     

Impacts of cage culture on physico-chemical and bacteriological water quality in Lake Volta,Ghana

The effects of cage fish farming on physico-chemical and bacteriological water quality in Lake Volta, Ghana, were investigated in 2013–2014. Farmed and unfarmed (control) areas of the lake were selected for monitoring. Nutrients, temperature, dissolved oxygen, conductivity, turbidity, pH, total coliforms, Pseudomonas and Vibrio spp. in the water were monitored monthly. Analyses of the water samples were carried out according to standard procedures. Physico-chemical quality of the water in both farm and control sites were within ranges typical of minimally impacted water and did not vary significantly between the two contrasting sites. The bacteriological analysis, however, revealed contamination of the lake water by fish farming. The bacterial counts at the farmed sites were significantly higher (p < 0.05) than those of the control sites, with figures at the farmed sites ranging from 132 to 1 708 cfu 100 ml?1 for total coliforms, 514 to 5 170 cfu 100 ml?1 Pseudomonas spp. and 14 to 516 cfu 100 ml?1 for Vibrio spp. The results suggested that cage fish farming has increased bacterial loads in the lake water, but has had minimal impact on its physico-chemical quality.     

Inventing the dynamo machine: the evolution of the F-type and V-type ATPases

The rotary proton- and sodium-translocating ATPases are reversible molecular machines present in all cellular life forms that couple ion movement across membranes with ATP hydrolysis or synthesis. Sequence and structural comparisons of F- and V-type ATPases have revealed homology between their catalytic and membrane subunits, but not between the subunits of the central stalk that connects the catalytic and membrane components. Based on this pattern of homology, we propose that these ATPases originated from membrane protein translocases, which, themselves, evolved from RNA translocases. We suggest that in these ancestral translocases, the position of the central stalk was occupied by the translocated polymer.     

Proton transfer in Azotobacter vinelandii ferredoxin I: entatic Lys84 operates as elastic counterbalance for the proton-carrying Asp15

In ferredoxin I from Azotobacter vinelandii, the reduction of a [3Fe-4S] iron-sulphur cluster is coupled with the protonation of the mu2S sulphur atom that is approx. 6 A away from the protein boundary. The recent study of the site-specific mutants of ferredoxin I led to the conclusion that a particular surface aspartic residue (Asp15) is solely responsible for the proton transfer to the mu2S atom by 'rapid penetrative excursions' (K. Chen, J. Hirst, R. Camba, C.A. Bonagura, C.D. Stout, B.K. Burgess, F.A. Armstrong, Nature 405 (2000) 814-817). In the same paper it has been reported that the replacement of Asp15 by glutamate led to the blockage of the enzyme, although glutamate, with its longer and more flexible side chain, should apparently do even better as a mobile proton carrier than aspartate. We tackled this puzzling incompetence of Glu15 by molecular dynamics simulations. It was revealed that the conformational alterations of Asp15 are energetically balanced by the straining of the nearby Lys84 side chain in wild-type ferredoxin I but not in the Asp15-->Glu mutant. Lys84 in ferredoxin I of A. vinelandii seems to represent the first case where the strained (entatic) conformation of a particular amino acid side chain could be directly identified in the ground state of an enzyme and assigned to a distinct mechanism of energy balance during the catalytic transition.     

Photosystem II of green plants: on the possible role of retarded protonic relaxation in water oxidation1

Photosystem II (PSII) of green plants and cyanobacteria uses energy of light to oxidize water and to produce oxygen. The available estimates of the oxidizing potential of P680+, the primary donor of PSII, yield value of about 1.15 V. Two main factors are suggested to add up and engender this high oxidizing potential, namely: (1) the electrostatic influence dominated by Arg-181 of the D2 subunit which elevates the oxidizing potential of P680+ up to 1 V, some 0.1 V above the Em value of a hydrogen-bonded chlorophyll a; and (2) the dynamic component of 0.10-0.15 V due to the experimentally demonstrated retarded protonic relaxation at the P680 site.     

Serious adverse events following treatment with ivermectin for onchocerciasis control: a review of reported cases

This paper presents a summary of reported cases of Serious Adverse Events (SAEs) following treatment with Mectizan^® (ivermectin, Merck, Sharpe &; Dohme) in onchocerciasis mass treatment programs from January 1, 1989 to December 31, 2001 through a passive surveillance system. A total of 207 SAE cases were reported out of approximately 165 million reported treatments delivered during the period under review, giving rise to a cumulative incidence of 1 reported SAE per 800,000 reported treatments. The mean age was 40 years and 70% of the cases were males. The mean time between ivermectin intake and onset of illness was 1 day. For 57% of the cases (n = 118), that was their first exposure to ivermectin. The majority of cases were reported from Cameroon (n = 176; 85%) with peaks in the incidence of SAE reporting in 1989–1991 and 1994–1995 when the program expanded to ivermectin-naïve populations. Fifty-five percent of the cases from Cameroon (i.e. 97 out of 176 cases) were encephalopathic and were reported from the central-southern region of the country; two-thirds of these cases were 'probable' or 'possible' cases of Loa loa encephalopathy temporally related to ivermectin treatment. Reporting bias may explain some but not all of the differences in SAE reporting between the 34 onchocerciasis-endemic countries that have, or have had, mass treatment programs. Further research is needed to understand the apparent clustering of encephalopathy cases in central-southern Cameroon since L. loa infection alone probably does not explain the increased incidence of this type of SAE from this region.     

Reduction and protonation of the secondary quinone acceptor of Rhodobacter sphaeroides photosynthetic reaction center: kinetic model based on a comparison of wild-type chromatophores with mutants carrying Arg-->Ile substitution at sites 207 and 217 in the L-subunit

After the light-induced charge separation in the photosynthetic reaction center (RC) of Rhodobacter sphaeroides, the electron reaches, via the tightly bound ubiquinone QA, the loosely bound ubiquinone Q(B) After two subsequent flashes of light, Q(B) is reduced to ubiquinol Q(B)H2, with a semiquinone anion Q-(B) formed as an intermediate after the first flash. We studied Q(B)H2 formation in chromatophores from Rb. sphaeroides mutants that carried Arg-->Ile substitution at sites 207 and 217 in the L-subunit. While Arg-L207 is 17 A away from Q(B), Arg-L217 is closer (9 A) and contacts the Q(B)-binding pocket. From the pH dependence of the charge recombination in the RC after the first flash, we estimated deltaG(AB), the free energy difference between the Q-(A)Q(B) and Q(A)Q-(B) states, and pK212, the apparent pK of Glu-L212, a residue that is only 4 A away from Q(B). As expected, the replacement of positively charged arginines by neutral isoleucines destabilized the Q-(B) state in the L217RI mutant to a larger extent than in the L207RI one. Also as expected, pK212 increased by approximately 0.4 pH units in the L207RI mutant. The value of pK212 in the L217RI mutant decreased by 0.3 pH units, contrary to expectations. The rate of the Q-(A)Q-(B)-->Q(A)Q(B)H2 transition upon the second flash, as monitored by electrometry via the accompanying changes in the membrane potential, was two times faster in the L207RI mutant than in the wild-type, but remained essentially unchanged in the L217RI mutant. To rationalize these findings, we developed and analyzed a kinetic model of the Q-(A)Q-(B)-->Q(A)Q(B)H2 transition. The model properly described the available experimental data and provided a set of quantitative kinetic and thermodynamic parameters of the Q(B) turnover. The non-electrostatic, 'chemical' affinity of the QB site to protons proved to be as important for the attracting protons from the bulk, as the appropriate electrostatic potential. The mutation-caused changes in the chemical proton affinity could be estimated from the difference between the experimentally established pK2J2 shifts and the expected changes in the electrostatic potential at Glu-L212, calculable from the X-ray structure of the RC. Based on functional studies, structural data and kinetic modeling, we suggest a mechanistic scheme of the QB turnover. The detachment of the formed ubiquinol from its proximal position next to Glu-L212 is considered as the rate-limiting step of the reaction cycle.     

Photosynthetic electrogenic events in native membranes of Chloroflexus aurantiacus. Flash-induced charge displacements in the acceptor quinone complex of the photosynthetic reaction center

Light-dependent reduction of cystine disulfide bonds results in activation of several of the enzymes of photosynthetic carbon metabolism within the chloroplast. Tertiary structure modeling suggests that the redox-sensitivity of the chloroplast malate dehydrogenase (EC 1.1.1.82) is due to disulfide crosslinking of the carbon substrate and nucleotide-binding domains. Consistent with this suggestion, introduction of Cys residues in opposition to one another on the two domains of the Escherichia coli enzyme results in redox-sensitivity [Muslin EH et al. (1995) Biophys J 68: 2218-2223]. We have now substituted Cys residues into the bacterial malate dehydrogenase (EC 1.1.1.37) in positions that correspond more exactly to those postulated to be responsible for the redox-sensitivity of the chloroplast enzyme. The introduction of one pair of Cys residues renders the enzyme redox-sensitive, but the introduction of the alternate pair does not. Energy minimization calculations suggest that the difference in redox-sensitivity is consistent with differences in the energy required for formation of the disulfide bond.     

Prevention of peroxidation of cardiolipin liposomes by quinol-based antioxidants

In mammalian mitochondria, cardiolipin molecules are the primary targets of oxidation by reactive oxygen species. The interaction of oxidized cardiolipin molecules with the constituents of the apoptotic cascade may lead to cell death. In the present study, we compared the effects of quinol-containing synthetic and natural amphiphilic antioxidants on cardiolipin peroxidation in a model system (liposomes of bovine cardiolipin). We found that both natural ubiquinol and synthetic antioxidants, even being introduced in micro- and submicromolar concentrations, fully protected the liposomal cardiolipin from peroxidation. The duration of their action, however, varied; it increased with the presence of either methoxy groups of ubiquinol or additional reduced redox groups (in the cases of rhodamine and berberine derivates). The concentration of ubiquinol in the mitochondrial membrane substantially exceeds the concentrations of antioxidants we used and would seem to fully prevent peroxidation of membrane cardiolipin. In fact, this does not happen: cardiolipin in mitochondria is oxidized, and this process can be blocked by amphiphilic cationic antioxidants (Y. N. Antonenko et al. (2008) Biochemistry (Moscow), 73, 1273–1287). We suppose that a fraction of mitochondrial cardiolipin could not be protected by natural ubiquinol; in vivo, peroxidation most likely threatens those cardiolipin molecules that, being bound within complexes of membrane proteins, are inaccessible to the bulky hydrophobic ubiquinol molecules diffusing in the lipid bilayer of the inner mitochondrial membrane. The ability to protect these occluded cardiolipin molecules from peroxidation may explain the beneficial therapeutic action of cationic antioxidants, which accumulate electrophoretically within mitochondria under the action of membrane potential.     

Proton slip in the ATP synthase of Rhodobacter capsulatus: induction, proton conduction, and nucleotide dependence

FOF1-ATP synthase converts two energetic "currencies" of the cell (ATP and protonmotive force, pmf) by coupling two rotary motors/generators. Their coupling efficiency is usually very high. Uncoupled proton leakage (slip) has only been observed in chloroplast enzyme at unphysiologically low nucleotide concentration. We investigated the properties of proton slip in chromatophores (sub-bacterial vesicles) from Rhodobacter capsulatus in the single-enzyme-per-vesicle mode. The membrane was energized by excitation with flashing light and the relaxation of the transmembrane voltage and pH difference was photometrically detected. We found that: (1) Proton slip occurred only at low nucleotide concentration (<1 microM) and after pre-illumination over several seconds. (2) Slip induction by pmf was accompanied by the release of approximately 0.25 mol ADP per mole of enzyme. There was no detectable detachment of F1 from FO. (3) The transmembrane voltage and the pH difference were both efficient in slip induction. Once induced, slip persisted for hours, and was only partially reverted by the addition of ADP or ATP (>1 microM). (4) There was no pmf threshold for the proton transfer through the slipping enzyme; slip could be driven both by voltage and pH difference. (5) The conduction was ohmic and weakly pH-dependent in the range from 5.5 to 9.5. The rate constant of proton transfer under slip conditions was 185 s(-1) at pH 8. Proton slip probably presents the free-wheeling of the central rotary shaft, subunit gamma, in an open structure of the (alphabeta)3 hexagon with no nucleotides in the catalytic sites.