Inhibition of Sulfate Respiration by 1,8-Dihydroxyanthraquinone and Other Anthraquinone Derivatives

Derivatives of 9,10-anthracenedione, or anthraquinone, were shown to inhibit respiratory sulfate reduction by pure cultures of sulfate-reducing bacteria, as well as by crude enrichment cultures. Structure-activity studies showed that an increasing degree of substitution of the anthraquinone nucleus resulted in increasing 50% inhibition (I(inf50)) values for sulfate respiration. Addition of charged ring substituents also resulted in an increase in the I(inf50) concentration. Experiments carried out with 1,8-dihydroxyanthraquinone demonstrated inhibition of hydrogen-dependent sulfate respiration but not hydrogen-dependent sulfite or thiosulfate respiration. Addition of pyruvate resulted in stimulation of sulfate-dependent hydrogen oxidation in the presence of the anthraquinone. These observations, together with a direct demonstration of uncoupling in French press vesicle preparations, suggest that the underlying mechanism of inhibition is uncoupling of ATP synthesis from electron transfer reactions. The low I(inf50) values for inhibition (0.5 to 10 (mu)M) and the relatively low general toxicity of anthraquinones suggest that these compounds may be useful for inhibition of sulfide generation in situations which are incompatible with the use of broadly toxic biocides.     

A carboxylic residue at the high-affinity, Mn-binding site participates in the binding of iron cations that block the site

The role of carboxylic residues at the high-affinity, Mn-binding site in the ligation of iron cations blocking the site [Biochemistry 41 (2000) 5854] was studied, using a method developed to extract the iron cations blocking the site. We found that specifically bound Fe(III) cations can be extracted with citrate buffer at pH 3.0. Furthermore, citrate can also prevent the photooxidation of Fe(II) cations by Y_Z. Participation of a COOH group(s) in the ligation of Fe(III) at the high-affinity site was investigated using 1-ethyl-3-[(3-dimethylamino)propyl] carbodiimide (EDC), a chemical modifier of carboxylic amino acid residues. Modification of the COOH groups inhibits the light-induced oxidation of exogenous Mn(II) cations by Mn-depleted photosystem II (PSII[−Mn]) membranes. The rate of Mn(II) oxidation saturates at ≥10 μM in PSII(−Mn) membranes and ≥500 μM in EDC-treated PSII (−Mn) samples. Intact PSII(−Mn) membranes have only one site for Mn(II) oxidation via Y_Z (dissociation constant, K_d = 0.64 μM), while EDC-treated PSII(−Mn) samples have two sites (K_d = 1.52 and 22 μM; the latter is the low-affinity site). When PSII(−Mn) membranes were incubated with Fe(II) before modifier treatment (to block the high-affinity site) and the blocking iron cations were extracted with citrate (pH 3.0) after modification, the membranes contained only one site (K_d = 2.3 μM) for exogenous Mn(II) oxidation by Y_Z radical. In this case, the rate of electron donation via Y_Z saturated at a Mn(II) concentration ≥15 μM. These results indicate that the carboxylic residue participating in Mn(II) coordination and the binding of oxidized manganese cations at the HA_Z site is protected from the action of the modifier by the iron cations blocking the HA_Z site. We concluded that the carboxylic residue (D1 Asp-170) participating in the coordination of the manganese cation at the HA_Z site (Mn4 in the tetranuclear manganese cluster [Science 303 (2004) 1831]) is also involved in the ligation of the Fe cation(s) blocking the high-affinity Mn-binding site.     

Photophysical behavior and assignment of the low-energy chlorophyll states in the CP43 proximal antenna protein of higher plant photosystem II

We have employed absorption, circular dichroism (CD), and persistent spectral hole-burning measurements at 1.7 K to study the photoconversion properties and exciton coupling of low-energy chlorophylls (Chls) in the CP43 proximal antenna light-harvesting subunit of photosystem II (PSII) isolated from spinach. These approximately 683 nm states act as traps for excitation energy in isolated CP43. They "bleach" at 683 nm upon illumination and photoconvert to a form absorbing in the range approximately 660-680 nm. We present new data that show the changes in the CD spectrum due to the photoconversion process. These changes occur in parallel with those in absorption, providing evidence that the feature undergoing the apparent bleach is a component of a weakly exciton-coupled system. From our photoconversion difference spectra, we assign four states in the Chl long-wavelength region of CP43, two of which are the known trap states and are both highly localized on single Chls. The other two states are associated with weak exciton coupling (maximally approximately 50 cm(-)(1)) to one of these traps. We propose a mechanism for photoconversion that involves Chl-protein hydrogen bonding. New hole-burning data are presented that indicate this mechanism is distinct to that for narrow-band spectral hole burning in CP43. We discuss the photophysical behavior of the Chl trap states in isolated CP43 compared to their behavior in intact PSII preparations. The latter represent a more intact, physiological complex, and we find no clear evidence that they exhibit the photoconversion process reported here.     

A carboxylic residue at the high-affinity, Mn-binding site participates in the binding of iron cations that block the site

The role of carboxylic residues at the high-affinity, Mn-binding site in the ligation of iron cations blocking the site [Biochemistry 41 (2000) 5854] was studied, using a method developed to extract the iron cations blocking the site. We found that specifically bound Fe(III) cations can be extracted with citrate buffer at pH 3.0. Furthermore, citrate can also prevent the photooxidation of Fe(II) cations by YZ. Participation of a COOH group(s) in the ligation of Fe(III) at the high-affinity site was investigated using 1-ethyl-3-[(3-dimethylamino)propyl] carbodiimide (EDC), a chemical modifier of carboxylic amino acid residues. Modification of the COOH groups inhibits the light-induced oxidation of exogenous Mn(II) cations by Mn-depleted photosystem II (PSII[-Mn]) membranes. The rate of Mn(II) oxidation saturates at > or = 10 microM in PSII(-Mn) membranes and > or = 500 microM in EDC-treated PSII (-Mn) samples. Intact PSII(-Mn) membranes have only one site for Mn(II) oxidation via YZ (dissociation constant, Kd = 0.64 microM), while EDC-treated PSII(-Mn) samples have two sites (Kd = 1.52 and 22 microM; the latter is the low-affinity site). When PSII(-Mn) membranes were incubated with Fe(II) before modifier treatment (to block the high-affinity site) and the blocking iron cations were extracted with citrate (pH 3.0) after modification, the membranes contained only one site (Kd = 2.3 microM) for exogenous Mn(II) oxidation by Y(Z)() radical. In this case, the rate of electron donation via YZ saturated at a Mn(II) concentration > or = 15 microM. These results indicate that the carboxylic residue participating in Mn(II) coordination and the binding of oxidized manganese cations at the HAZ site is protected from the action of the modifier by the iron cations blocking the HAZ site. We concluded that the carboxylic residue (D1 Asp-170) participating in the coordination of the manganese cation at the HAZ site (Mn4 in the tetranuclear manganese cluster [Science 303 (2004) 1831]) is also involved in the ligation of the Fe cation(s) blocking the high-affinity Mn-binding site.     

Hydrogen photoproduction by nutrient-deprived Chlamydomonas reinhardtii cells immobilized within thin alginate films under aerobic and anaerobic conditions

A new technique for immobilizing H2-photoproducing green algae within a thin (<400 microm) alginate film has been developed. Alginate films with entrapped sulfur/phosphorus-deprived Chlamydomonas reinhardtii, strain cc124, cells demonstrate (a) higher cell density (up to 2,000 microg Chl mL(-1) of matrix), (b) kinetics of H2 photoproduction similar to sulfur-deprived suspension cultures, (c) higher specific rates (up to 12.5 micromol mg(-1) Chl h(-1)) of H2 evolution, (d) light conversion efficiencies to H2 of over 1% and (e) unexpectedly high resistance of the H2-photoproducing system to inactivation by atmospheric O2. The algal cells, entrapped in alginate and then placed in vials containing 21% O2 in the headspace, evolved up to 67% of the H2 gas produced under anaerobic conditions. The results indicate that the lower susceptibility of the immobilized algal H2-producing system to inactivation by O2 depends on two factors: (a) the presence of acetate in the medium, which supports higher rates of respiration and (b) the capability of the alginate polymer itself to effectively separate the entrapped cells from O2 in the liquid and headspace and restrict O2 diffusion into the matrix. The strategy presented for immobilizing algal cells within thin polymeric matrices shows the potential for scale-up and possible future applications.     

Cytotoxic potency of trialkyltins to C6 glioma cells <Emphasis Type="Italic">in vitro</Emphasis>: impact of exposure conditions

Because of a possible role of astrocytes in trialkyltin-induced neurotoxicity in vivo various studies have been performed using cultures of astrocytes or glioma cells in vitro. With respect to cytotoxic potencies of trialkyltins these studies gave rather divergent results. Therefore the aim of the present study was to clarify whether variations of experimental conditions could be responsible for the differences of the cytotoxic activities of trimethyltin (TMT), triethyltin (TET) and tributyltin (TBT). Experiments were performed with rat C6 glioma cells. Toxicity was determined by measuring the reduction of the cell protein content. Cultures of proliferating and growth-arrested cells did not differ in their sensitivity. Exposure duration (1–72 h) had a strong but differing influence on the cytotoxic potency of the trialkyltins. After short exposure times the potencies differed largely (TMT < TET < TBT), whereas they became more and more similar with increasing exposure duration. The potency–time relationships for TMT and TET could be described by the equation: EC₅₀ = k x t ^–n, while for TBT an incipient value (EC_50,) had to be included: EC₅₀ = EC ₅₀ + k x t ^–n. Addition of serum albumin to the culture medium decreased the cytotoxic potency of the trialkyltins. However, the impact of protein binding on their bioavailability was relatively low. The cytotoxic potency of the alkyltins was not dependent on the concentration of C6 cells. Taken together, neither differences in exposure conditions nor in the proliferative status of the cells are sufficient to account for the discrepancies in published results for trialkyltin cytotoxicity to astrocytes. Instead they may – at least partially – be explained by differing sensitivities of the endpoints used. Furthermore, C6 glioma cells respond considerably more sensitively to trialkytins than primary astrocytes, which questions their applicability as models for astrocyte toxicity.