The kinetics of the degradation of chloroform and benzene in anaerobic sediment from the River Rhine

In anaerobic methanogenic sediment microcosms¹⁴C labelled chloroform was degraded mainly to carbon dioxide. At a concentration of 4 g.l^–1 the mineralization followed first order kinetics with a half life of 12 days at 10°C and 2.6 days at 20°C. At a concentration of 400 g.l^–1 the mineralization rate increased with time and followed logarithmic kinetics with a _max of 0.02.d^–1 at 10°C. The logarithmic kinetics can be explained by growth of the bacteria on the higher concentration of chloroform with a generation time of 35 days. Shaking and oxygenation did not inhibit the mineralization of chloroform, probably because of bacterial consumption of the dissolved oxygen. ¹⁴C labelled benzene was mineralized only for a small percentage to¹⁴C labelled carbon dioxide while other, not acid extractable, degradation products were formed. Under anaerobic conditions after one day when 5% of the benzene was degraded to carbon dioxide, the mineralization ceased, while the disappearance of benzene proceeded. With air in the headspace of the incubation bottles 25% of the benzene was mineralized to carbon dioxide. The anaerobic degradation of benzene at a concentration of 100 .l^–1 showed similar kinetics as the degradation at 1 g.l^–1. Hence no adaptation of the microflora in the sediment occurred during the 63 days of the experiment at 100 g.l^–1.     

Reductive dechlorination of 3-chlorobenzoate is coupled to ATP production and growth in an anaerobic bacterium,strain DCB-1

Thermodynamic data that the reductive dechlorination of 3-chlorobenzoate is exergonic have led to the hypothesis that this reaction yields biologically useful energy. This hypothesis was tested with strain DCB-1, a dehalogenating bacterium. The organism was grown under strictly anaerobic conditions in vitamin-amended mineral medium with formate plus acetate as electron donor and 3-chlorobenzoate as electron acceptor. The cell yield increased stoichiometrically to the amount of 3-chlorobenzoate dechlorinated. No growth was observed in the absence of 3-chlorobenzoate, or when 3-chlorobenzoate was replaced by benzoate. To obtain further evidence on that energy is derived from dechlorination, 3-chlorobenzoate was added to starved cells. This amendment resulted in an increase in the ATP level of the cells at 10 nmol per mg protein versus 3 nmol per mg protein in non-amended controls. These data indicate that the reductive dehalogenation of chlorinated aromatic compounds can be coupled to a novel type of chemotrophy.     

A simple method for the determination of reduction potentials in heme proteins

We describe a simple method for the determination of heme protein reduction potentials. We use the method to determine the reduction potentials for the PAS-A domains of the regulatory heme proteins human NPAS2 (E_m = −115 mV ± 2 mV, pH 7.0) and human CLOCK (E_m = −111 mV ± 2 mV, pH 7.0). We suggest that the method can be easily and routinely applied to the determination of reduction potentials across the family of heme proteins.     

Redox potentials of the oriented film of the wild-type, the E194Q-, E204Q- and D96N-mutated bacteriorhodopsins

The redox potentials of the oriented films of the wild-type, the E194Q-, E204Q- and D96N-mutated bacteriorhodopsins (bR), prepared by adsorbing purple membrane (PM) sheets or its mutant on a Pt electrode, have been examined. The redox potentials (V) of the wild-type bR were −470 mV for the 13-cis configuration of the retinal Shiff base in bR and −757 mV for the all-trans configuration in H₂O, and −433 mV for the 13-cis configuration and −742 mV for the all-trans configuration in D₂O. The solvent isotope effect (ΔV=V(D₂O)−V(H₂O)), which shifts the redox potential to a higher value, originates from the cooperative rearrangements of the extensively hydrogen-bonded water molecules around the protonated CN part in the retinal Schiff base. The redox potential of bR was much higher for the 13-cis configuration than that for the all-trans configuration. The redox potentials for the E194Q mutant in the extracellular region were −507 mV for the 13-cis configuration and −788 mV for the all-trans configuration; and for the E204Q mutant they were −491 mV for the 13-cis configuration and −769 mV for the all-trans configuration. Replacement of the Glu¹⁹⁴ or Glu²⁰⁴ residues by Gln weakened the electron withdrawing interaction to the protonated CN bond in the retinal Schiff base. The E204 residue is less linked with the hydrogen-bonded network of the proton release pathway compared with E194. The redox potentials of the D96N mutant in the cytoplasmic region were −471 mV for the 13-cis configuration and −760 mV for the all-trans configuration which were virtually the same as those of the wild-type bR, indicating that the D to N point mutation of the 96 residue had no influence on the interaction between the D96 residue and the CN part in the Schiff base under the light-adapted condition. The results suggest that the redox potential of bR is closely correlated to the hydrogen-bonded network spanning from the retinal Schiff base to the extracellular surface of bR in the proton transfer pathway.     

Reductive deamination as a new step in the anaerobic microbial degradation of halogenated anilines

In this paper we report the isolation and characterization of an anaerobic enrichment culture as well as of a Rhodococcus sp. strain 2 capable of degrading 3,4-dihaloanilines under nitrate reducing conditions. Using mass spectrometry several of the intermediates formed in the process of 3,4-dichloroaniline conversion were identified. Most interesting is the observation of reductive deamination and the formation of 1,2-dichlorobenzene as one of the intermediates. Using 19F NMR and fluorinated 3,4-dihaloaniline model substrates it was corroborated that reductive deamination of the anilines to give dihalobenzene intermediates represents a new initial step in the anaerobic microbial degradation of these halogenated anilines.     

Redox properties of the Fe3+/Fe2+ couple in Arthromyces ramosus class II peroxidase and its cyanide adduct

The thermodynamics of the one-electron reduction of the ferric heme in free and cyanide-bound Arthromyces ramosus peroxidase (ARP), a class II plant peroxidase, were determined through spectro-electrochemical experiments. The data were compared with those for class III horseradish peroxidase C (HRP) and its cyanide adduct, and were interpreted in terms of ligand binding features, electrostatic effects and solvent accessible surface area of the heme group and of catalytically relevant residues in the heme distal site. The values for free and cyanide-bound ARP (−0.183 and −0.390 V, respectively, at 25 °C and pH 7) are higher than those for HRP and HRP-CN. ARP features an enthalpic stabilization of the ferrous state and a remarkably negative reduction entropy, which are both unprecedented for heme peroxidases. Once the compensatory contributions of solvent reorganization are partitioned from the measured reduction enthalpy, the resulting protein-based value for ARP turns out to be less positive than that for HRP by +10 kJ mol⁻¹. The smaller stabilization of the oxidized heme in ARP most probably results from the less pronounced anionic character of the proximal histidine, and the decreased polarity in the heme distal site as compared with HRP, as indicated by the X-ray structures. The surprisingly negative value for ARP is the result of peculiar reduction-induced solvent reorganization effects.     

Evaluations of reduction potential data in relation to coupling, kinetics and function

 This commentary addresses the effect and measurement of time-dependent contributions to reduction potentials. Reduction potentials form the basis for many quantitative or semi-quantitative judgements in biological redox chemistry. However, since data are obtained under an assumption of equilibrium being established, their relevance to biological functions requires consideration of the kinetics of the subprocesses that contribute to or influence the overall free energy change. Initial and final states effective in rapid and complex biological functions may differ considerably from those analysed after slow equilibration in a sample tube. A shortcoming of traditional potentiometric measurements is that the time domain is not probed. Voltammetry, a technique that has been much less widely applied in biological chemistry than in chemistry, examines redox transformations in both potential and time domains, and may enable a more realistic picture to be derived. Received, accepted: 26 November 1996     

Equilibrium and kinetic measurements of the redox potentials of cytochromes c2 in vitro and in vivo

The equilibrium oxidation-reduction mipoint potential (E_m) of isolated Rhodopseudomonas sphaeroides cytochrome c₂ exhibits a pH-dependent behavior which can be ascribed to a pK on the oxidized form at pH 8.0 (Pettigrew et al. (1975) Biochim. Biophys. Acta 430, 197–208). However, as with mammalian cytochrome c (Brandt, K.G., Parks, P.C., Czerlinski, G.H. and Hess, G.P. (1966) J. Biol. Chem. 241, 4180–4185) this pK can more properly be attributed to the combination of a pK beyond pH 11, and a slow conformational change of the ferricytochrome. This has been demonstrated by resolving the E_m of cytochrome c₂ before and after the conformational change. The E_m of the unaltered form is essentially pH independent between pH 7 and 11.5, and the lower equilibrium E_m is due solely to the conformational change. In vivo the conformational change is prevented by the binding of the cytochrome c₂ to the photochemical reaction center, and the cytochrome exhibits an essentially pH-independent E_m from pH 5 to 11. The alkaline transition thus has little physiological significance, and it is unlikely that the redox reactions of cytochrome c₂ in vivo involve protons.     

Redox biology in normal cells and cancer: Restoring function of the redox/Fyn/c-Cbl pathway in cancer cells offers new approaches to cancer treatment

This review discusses a unique discovery path starting with novel findings on redox regulation of precursor cell and signaling pathway function and identification of a new mechanism by which relatively small changes in redox status can control entire signaling networks that regulate self-renewal, differentiation, and survival. The pathway central to this work, the redox/Fyn/c-Cbl (RFC) pathway, converts small increases in oxidative status to pan-activation of the c-Cbl ubiquitin ligase, which controls multiple receptors and other proteins of central importance in precursor cell and cancer cell function. Integration of work on the RFC pathway with attempts to understand how treatment with systemic chemotherapy causes neurological problems led to the discovery that glioblastomas (GBMs) and basal-like breast cancers (BLBCs) inhibit c-Cbl function through altered utilization of the cytoskeletal regulators Cool-1/βpix and Cdc42, respectively. Inhibition of these proteins to restore normal c-Cbl function suppresses cancer cell division, increases sensitivity to chemotherapy, disrupts tumor-initiating cell (TIC) activity in GBMs and BLBCs, controls multiple critical TIC regulators, and also allows targeting of non-TICs. Moreover, these manipulations do not increase chemosensitivity or suppress division of nontransformed cells. Restoration of normal c-Cbl function also allows more effective harnessing of estrogen receptor-α (ERα)-independent activities of tamoxifen to activate the RFC pathway and target ERα-negative cancer cells. Our work thus provides a discovery strategy that reveals mechanisms and therapeutic targets that cannot be deduced by standard genetics analyses, which fail to reveal the metabolic information, isoform shifts, protein activation, protein complexes, and protein degradation critical to our discoveries.     

Disruption of the H-bond network in the main access channel of catalase-peroxidase modulates enthalpy and entropy of Fe(III) reduction

Catalase-peroxidases are the only heme peroxidases with substantial hydrogen peroxide dismutation activity. In order to understand the role of the redox chemistry in their bifunctional activity, catalatically-active and inactive mutant proteins have been probed in spectroelectrochemical experiments. In detail, wild-type KatG from Synechocystis has been compared with variants with (i) disrupted KatG-typical adduct (Trp122-Tyr249-Met275), (ii) mutation of the catalytic distal His123-Arg119 pair, and (iii) altered accessibility to the heme cavity (Asp152, Ser335) and modified charge at the substrate channel entrance (Glu253). A valuable insight into the mechanism of reduction potential (E°′) modulation in KatG has been obtained from the parameterization of the corresponding enthalpic and entropic components, determined from the analysis of the temperature dependence of E°′. Moreover, model structures of ferric and ferrous Synechocystis KatG have been computed and used as reference to analyze and discuss the experimental data. The results, discussed by reference to published resonance Raman data on the strength of the proximal iron-imidazole bond and catalytic properties, demonstrate that E°′ of the Fe(III)/Fe(II) couple is not strongly correlated with the bifunctional activity. Besides the importance of an intact Trp-Tyr-Met adduct, it is the architecture of the long and constricted main channel that distinguishes KatGs from monofunctional peroxidases. An ordered matrix of oriented water dipoles is important for H₂O₂ oxidation. Its disruption results in modification of enthalpic and entropic contributions to E°′ that reflect reduction-induced changes in polarity, electrostatics, continuity and accessibility of solvent to the metal center as well as alterations in solvent reorganization.     

Mutational and spectroscopic studies of the significance of the active site glutamine to metal ion specificity in superoxide dismutase

We are addressing the puzzling metal ion specificity of Fe- and Mn-containing superoxide dismutases (SODs) [see C.K.Vance, A.-F. Miller, J. Am. Chem. Soc. 120(3) (1998) 461–467]. Here, we test the significance to activity and active site integrity of the Gln side chain at the center of the active site hydrogen bond network. We have generated a mutant of MnSOD with the active site Gln in the location characteristic of Fe-specific SODs. The active site is similar to that of MnSOD when Mn²⁺, Fe³⁺ or Fe²⁺ are bound, based on EPR and NMR spectroscopy. However, the mutant’s Fe-supported activity is at least 7% that of FeSOD, in contrast to Fe(Mn)SOD, which has 0% of FeSOD’s activity. Thus, moving the active site Gln converts Mn-specific SOD into a cambialistic SOD and the Gln proves to be important but not the sole determinant of metal-ion specificity. Indeed, subtle differences in the spectra of Mn²⁺, Fe³⁺ and ¹H in the presence of Fe²⁺ distinguish the G77Q, Q146A mut-(Mn)SOD from WT (Mn)SOD, and may prove to be correlated with metal ion activity. We have directly observed the side chain of the active site Gln in Fe²⁺SOD and Fe²⁺(Mn)SOD by ¹⁵N NMR. The very different chemical shifts indicate that the active site Gln interacts differently with Fe²⁺ in the two proteins. Since a shorter distance from Gln to Fe and stronger interaction with Fe correlate with a lower E_m in Fe(Mn)SOD, Gln has the effect of destabilizing additional electron density on the metal ion. It may do this by stabilizing OH⁻ coordinated to the metal ion.     

Effect of the energy source on the NADH/NAD ratio and on pyruvate catabolism in anaerobic chemostat cultures of Enterococcus faecalis NCTC 775

Abstract: Enterococcus faecalis was grown under anaerobic conditions in chemostat cultures on energy sources with different degress of reduction (i.e. mannitol, glucose, pyruvate) at various culture pH values. Intracellular NADH/NAD ratios were measured and were found to be influenced both by the nature of the energy source and by the culture pH value. Highest ratios were found with mannitol as energy source and with high culture pH values. A role for the redox potential of the NADH/NAD couple as a regulatory effector is suggested by a correlation of the redox potential with the in vivo distribution of the carbon flux between pyruvate formate lyase and the pyruvate dehydrogenase complex.     

Contribution of 13C-NMR spectroscopy to the elucidation of pathways of propionate formation and degradation in methanogenic environments

Propionate is an important intermediate in the anaerobic degradation of complex organic matter to methane and carbon dioxide. The metabolism of propionate-forming and propionate-degrading bacteria is reviewed here. Propionate is formed during fermentation of polysaccharides, proteins and fats. The study of the fate of 13C-labelled compounds by nuclear magnetic resonance (NMR) spectroscopy has contributed together with other techniques to the present knowledge of the metabolic routes which lead to propionate formation from these substrates. Since propionate oxidation under methanogenic conditions is thermodynamically difficult, propionate often accumulates when the rates of its formation and degradation are unbalanced. Bacteria which are able to degrade propionate to the methanogenic substrates acetate and hydrogen can only perform this reaction when the methanogens consume acetate and hydrogen efficiently. As a consequence, propionate can only be degraded by obligatory syntrophic consortia of microorganisms. NMR techniques were used to study the degradation of propionate by defined and less defined cultures of these syntrophic consortia. Different types of side-reactions were reported, like the reductive carboxylation to butyrate and the reductive acetylation to higher fatty acids.     

Redox titration of electron acceptor Q and the plastoquinone pool in Photosystem II

The primary photochemical quencher Q and the secondary electron acceptor pool in Photosystem II have been titrated. We used particles of Scenedesmus mutant No. 8 that lack System I and allowed the system to equilibrate with external redox mediators in darkness prior to measurement of the fluorescence rise curve.The titration of Q, as indicated by the dark level of F_i, occurs in two discrete steps. The high-potential component (Q_h) has a midpoint potential of +68 mV (pH 7.2) and accounts for ～67% of Q. The pH sensitivity of the midpoint potential is ?60 mV, indicating the involvement of 1 $\text{H}{}^{\mathrm{+}}\text{e}$. The low-potential component (Q₁) accounts for the remaining 33% of Q and shows a midpoint potential near?300 mV (pH 7.2).The plastoquinone pool, assayed as the half-time of the fluorescence rise curve, titrates as a single component with a midpoint potential 30–40 mV more oxidizing than that of Q_h, i.e., at 106 mV (pH 7.2). The E_m shows a pH sensitivity of ?60 mV/pH unit, indicating the involvement of 1 $\text{H}{}^{\mathrm{+}}\text{e}$. The observation that all 12–14 electron equivalents in the pool titrate as a single component indicates that the heterogeneity otherwise observed in the secondary acceptor system is a kinetic rather than a thermodynamic property.Illumination causes peculiar, and as yet unclarified, changes of both Q and the secondary pool under anaerobic conditions that are reversed by oxygen.     

ATP and NADPH as the driving force of carbon reduction in leaves in relation to thylakoid energization by light

Carbon assimilation of spinach (Spinacia oleracea L.) leaves was measured in the presence of 2000l· l^–1CO₂ and 2% O₂ in the gas phase to suppress photorespiratory reactions and to reduce stomatal diffusion resistance. Simultaneously, membrane parameters such as modulated chlorophyll fluorescence, oxidation of P₇₀₀ in the reaction centre of photosystem I, and apparent changes in absorbance at 535 nm were recorded. After light-regulated enzymes were activated at a high irradiance, illumination was changed. About 3 min later (to maintain the previous activation state of enzymes), leaves were shock-frozen and freeze-dried. Chloroplasts were isolated nonaqueously and analysed for ATP, ADP, inorganic phosphate, NADPH and NADP. Observations made under the chosen conditions differed in some important aspects from those commonly observed when leaves are illuminated in air. (i) Not only assimilation, but also the phosphorylation potential [ATP]/([ADP]·[Pi]) increased hyperbolically with irradiance towards saturation. In contrast, the ratio of NADPH to NADP did not change much as irradiances increased from low to high photon flux densities. When ATP, the phosphorylation potential and the assimilatory force, F_A (the product of phosphorylation potential and NADPH/NADP ratio), were plotted against assimilation, ATP increased relatively less than assimilation, whereas the phosphorylation potential increased somewhat more steeply than assimilation did. A linear relationship existed between assimilation and F_A at lower irradiances. The assimilatory force F_A increased more than assimilation did when irradiances were very high. Differences from previous observations, where F_A was under some conditions higher at low than at high rates of carbon assimilation, are explained by differences in flux resistances caused not only by stomatal diffusion resistance but also by differences in the activity of light-regulated enzymes, (ii) The relationship between P₇₀₀ oxidation and a fast absorption change with a maximum close to 520 nm on one hand and carbon assimilation on the other hand was largely linear under the specific conditions of the experiments. A similar linear relationship existed also between the quantum efficiency of electron flow through photosystem II and the quantum efficiency of photosystem I electron transport. (iii) Whereas the increase in non-photochemical fluorescence quenching, q_N, was similar to the increase in assimilation, the relationship between light scattering and assimilation was distinctly sigmoidal. Light scattering appeared to be a better indicator of control of photosystem II activity under excessive irradiation than q_N. (iv) The results are discussed in relation to the relative significance of chloroplast levels of ATP and NADPH and of the assimilatory force F_A in driving carbon assimilation. From the observations, the proton/electron (H⁺/e^–) ratio of linear electron transport is suggested to be 3 and the H⁺/ATP ratio to be 4 in leaves. An H⁺/e^– ratio of 3 implies the existence of an obligatory Q-cycle in leaves.Abbreviations F_A assimilatory force - F_o fluorescence after long dark adaptation - F_m maximum fluorescence level - F_s steady-state fluorescence - PGA 3-phosphoglycerate - PFD photon flux density - P₇₀₀ (P₇₀₀+) electron-donor pigment in the reaction center of PSI (its oxidized form) - Q_A primary quinone acceptor of PSII - q_P photochemical quenching - q_N non-photochemical quenching - _PSII relative quantum efficiency of energy conversation at the level of photosystem II - _PSI relative quantum efficiency of photosystem II This research was supported by the Sonderforschungsbereich 251 of the University of Würzburg and the Stiftung Volkswagenwerk. U.G. is a member of the Graduate College of the Julius-von-Sachs Institut für Biowissenschaften, University of Würzburg, being on leave from Tartu University, Tartu, Estonia. The authors are grateful to Prof. A. Laisk, Chair of Plant Physiology, Tartu University, for stimulating discussions.     

Functional anaerobic electron transport linked to the reduction of nitrate and fumarate in membranes from Escherichia coli as demonstrated by quenching of atebrin fluorescence.

Measurements were made of energy-dependent quenching of atebrin fluorescence in membrane particles prepared from Escherichia coli grown anaerobically with glycerol as carbon source in the presence of either nitrate or fumarate. It is concluded that this technique can be used to study the functional organization of the anaerobic proton-translocating electron-transport chains that use nitrate or fumarate as terminal electron acceptor.     

Regulation of Claspin degradation by the ubiquitin-proteosome pathway during the cell cycle and in response to ATR-dependent checkpoint activation

Claspin is involved in ATR-dependent activation of Chk1 during DNA replication and in response to DNA damage. We show that degradation of Claspin by the ubiquitin-proteosome pathway is regulated during the cell cycle. Claspin is stabilized in S-phase but is abruptly degraded in mitosis and is absent from early G(1) cells in which the phosphorylation of Chk1 by ATR is abrogated. In response to hydroxyurea, UV or aphidicolin, Claspin is phosphorylated in the Chk1-binding domain and its protein levels are increased in an ATR-dependent manner. Thus, the Chk1 pathway is regulated through both phosphorylation of Claspin and its controlled degradation.     

Using multi-target clustering trees as a tool to predict biological water quality indices based on benthic macroinvertebrates and environmental parameters in the Chaguana watershed (Ecuador)

Macroinvertebrates were sampled in the Chaguana river basin in SW Ecuador in the wet season (March) and the dry season (September) of 2005 and 2006. To assess the robustness of several biological indicators, correlations were calculated between both years and between the wet and the dry season. In addition, it was tested if the indices gave significantly different results for sites with a bad, poor, moderate and good ecological water quality. Composition measures performed poorly in most cases, however, abundance, diversity and richness measures often performed better and tolerance measures, the so-called biotic indices, performed very well, even indices developed for temperate regions. By using pruned multitarget clustering trees, it was possible to predict several well-performing ecological water quality indices simultaneously on the basis of the occurring key macroinvertebrate taxa or, alternatively, on the basis of key environmental variables. In contrast to unpruned trees, which resulted in complex trees that were difficult to interpret and performed inferiorly, pruning resulted in transparent trees. Water quality indices scored high when Hydropsychidae were present and even higher when in addition also Megapodagrionidae were present. When no Hydropsychidae nor Libellulidae were present, the indices reached the lowest scores. However, this model based on key taxa occurrences did not perform well during validation. Water quality indices scored higher with increasing dissolved oxygen concentrations and a strong current velocity. The latter model based on environmental variables also performed well during validation. In the presented study, the ecological water quality could thus be accurately predicted solely on the basis of dissolved oxygen concentration and current velocity. It can therefore be concluded that multitarget clustering trees can be easily used as a practical tool for cost-effective decision support by water quality managers.