Electrochemical and electron transfer investigations of copper proteins

A study of the pH and temperature dependence of the redox potentials of azurins from five species of bacteria has been performed. The variations in the potentials with pH have been interpreted in terms of electrostatic interactions between the copper site and titrating histidine residues, including the effects of substitutions in the amino acid sequences of the proteins on the electrostatic interactions. A comparison of the observed pH dependences with predictions based on histidine pK_a values known for Pseudomonas aeruginosa (Pae), Alcaligenes denitrificans (Ade), and Alcaligenes faecalis (Afa) azurins indicates that the Pae and Ade redox potentials exhibit pH dependences in line with electrostatic arguments, while Afa azurin exhibits more complex behavior. Redox enthalpies and entropies for four of the azurins at low and high pH values have also been obtained. Based on these results in conjuction with the variable pH experiments, it appears that Bordetella bronchiseptica azurin may undergo a more substantial conformational change with pH than has been observed for other species of azurin.

The temperature dependence of the redox potential of bovine erythrocyte superoxide dismutase (SOD) has been determined at pH 7.0, with potassium ferricyanide as the mediator. The following thermodynamic parameters have been obtained (T = 25°C): E°' = 403±5 mV vs. NHE, ΔG°' = -9.31 kcal/mol, ΔH°' = -21.4 kcal/mol, ΔS°' = -40.7 eu, ΔS°'_(rc) = -25.1 eu. It is apparent from these results that ΔH°', rather than ΔS°', is the dominant factor in establishing the high redox potential of SOD. The large negative enthalpy of reduction may also reflect the factors which give SOD its high specificity toward reduction and oxidation by superoxide.

Physical characterization of the rack effect and hydrogen bond networks in blue copper proteins

A summary of previous research is presented that indicates that the purpose of a blue copper protein's fold and hydrogen bond network, aka, the rack effect, enforce a copper(II) geometry around the copper(I) ion in the metal site. In several blue copper proteins, the C-terminal histidine ligand becomes protonated and detaches from the copper in the reduced forms. Mutants of amicyanin from Paracoccus denitrificans were made to alter the hydrogen bond network and quantify the rack effect by pK_a shifts.

The pK_a's of mutant amicyanins have been measured by pH-dependent electrochemistry. P94F and P94A mutations loosen the Northern loop, allowing the reduced copper to adopt a relaxed conformation: the ability to relax drives the reduction potentials up. The measured potentials are 265 (wild type), 380 (P94A), and 415 (P94F) mV vs. NHE. The measured pK_a's are 7.0 (wild type), 6.3 (P94A), and 5.0 (P94F). The additional hydrogen bond to the thiolate in the mutants is indicated by a red-shift in the blue copper absorption and an increase in the parallel hyperfine splitting in the EPR spectrum. This hydrogen bond is invoked as the cause for the increased stability of the C-terminal imidazole.

Melting curves give a measure of the thermal stability of the protein. A thermodynamic intermediate with pH-dependent reversibility is revealed. Comparisons with the electrochemistry and apoamicyanin suggest that the intermediate involves the region of the protein near the metal site. This region is destabilized in the P94F mutant; coupled with the evidence that the imidazole is stabilized under the same conditions confirms an original concept of the rack effect: a high energy configuration is stabilized at a cost to the rest of the protein.

Design, Synthesis, and Study of Novel Platforms for Iron-N2 Chemistry and Photoinduced, Copper-mediated C-N Bond Formation

Several new ligand platforms designed to support iron dinitrogen chemistry have been developed. First, we report Fe complexes of a tris(phosphino)alkyl (CP^iPr₃) ligand featuring an axial carbon donor intended to conceptually model the interstitial carbide atom of the nitrogenase iron-molybdenum cofactor (FeMoco). It is established that in this scaffold, the iron center binds dinitrogen trans to the C_alkyl anchor in three structurally characterized oxidation states. Fe-C_alkyl lengthening is observed upon reduction, reflective of significant ionic character in the Fe-C_alkyl interaction. The anionic (CP^iPr₃)FeN₂^- species can be functionalized by a silyl electrophile to generate (CP^iPr₃)Fe-N₂SiR₃. This species also functions as a modest catalyst for the reduction of N₂ to NH₃. Next, we introduce a new binucleating ligand scaffold that supports an Fe(μ-SAr)Fe diiron subunit that coordinates dinitrogen (N₂-Fe(μ-SAr)Fe-N₂) across at least three oxidation states (Fe^IIFe^II, Fe^IIFe^I, and Fe^IFe^I). Despite the sulfur-rich coordination environment of iron in FeMoco, synthetic examples of transition metal model complexes that bind N₂ and also feature sulfur donor ligands remain scarce; these complexes thus represent an unusual series of low-valent diiron complexes featuring thiolate and dinitrogen ligands. The (N₂-Fe(μ-SAr)Fe-N₂) system undergoes reduction of the bound N₂ to produce NH₃ (~50% yield) and can efficiently catalyze the disproportionation of N₂H₄ to NH₃ and N₂. The present scaffold also supports dinitrogen binding concomitant with hydride as a co-ligand. Next, inspired by the importance of secondary-sphere interactions in many metalloenzymes, we present complexes of iron in two new ligand scaffolds ([SiP^NMe₃] and [SiP^iPr₂P^NMe]) that incorporate hydrogen-bond acceptors (tertiary amines) which engage in interactions with nitrogenous substrates bound to the iron center (NH₃ and N₂H₄). Cation binding is also facilitated in anionic Fe(0)-N₂ complexes. While Fe-N₂ complexes of a related ligand ([SiP^iPr₃]) lacking hydrogen-bond acceptors produce a substantial amount of ammonia when treated with acid and reductant, the presence of the pendant amines instead facilitates the formation of metal hydride species.

Additionally, we present the development and mechanistic study of copper-mediated and copper-catalyzed photoinduced C-N bond forming reactions. Irradiation of a copper-amido complex, ((m-tol)₃P)₂Cu(carbazolide), in the presence of aryl halides furnishes N-phenylcarbazole under mild conditions. The mechanism likely proceeds via single-electron transfer from an excited state of the copper complex to the aryl halide, generating an aryl radical. An array of experimental data are consistent with a radical intermediate, including a cyclization/stereochemical investigation and a reactivity study, providing the first substantial experimental support for the viability of a radical pathway for Ullmann C-N bond formation. The copper complex can also be used as a precatalyst for Ullmann C-N couplings. We also disclose further study of catalytic C_alkyl-N couplings using a CuI precatalyst, and discuss the likely role of [Cu(carbazolide)₂]^- and [Cu(carbazolide)₃]^- species as intermediates in these reactions.

Finally, we report a series of four-coordinate, pseudotetrahedral P₃Fe^II-X complexes supported by tris(phosphine)borate ([PhBP₃Fe^R]^-) and phosphiniminato X-type ligands (-N=PR'₃) that in combination tune the spin-crossover behavior of the system. Low-coordinate transition metal complexes such as these that undergo reversible spin-crossover remain rare, and the spin equilibria of these systems have been studied in detail by a suite of spectroscopic techniques.

Environmental effects of drought and abstractions of River Wharfe fisheries, summer 1997

This investigation was carried out to provide information on fish stocks and angling activity during 1997 in relation to the drought and, in particular, flows as influenced by Time Limited Licences. These abstractions will be for review in 1999. This report extends and updates the data presented for 1996. Fish population surveys (including eels) were undertaken on the main river and selected tributaries. Angler caught brown trout were examined, angler catch data have been reviewed, and observations by Environment Agency fisheries staff collated. It appeared that in River Wharfe both the fish populations and individual fish appeared to be in good condition and limited changes had occurred since the 1996 survey.

Electrofishing survey of the River Calder and its tributaries - April 1978.

An electrofishing survey was undertaken in the period April 24th - 28th as part of the detailed biological study of the Calder Catchment being carried out by this department in early 1978. The sites to be fished were selected to indicate broadly the fishing status of the major sections of the main river and its larger tributaries and by no means intended to describe the fishing status of the whole catchment in detail. The results on the distributions of fish species are discussed, in general, in relation to the major polluting influences on the catchment. The results of the second survey are given and discussed separately in Appendix I.

Growth and survival of sockeye salmon (Oncorhynchus nerka) from Karluk Lake and River, Alaska, in relation to climatic and oceanic regimes and indices, 1922–2000

We examined whether the relationship between climate and salmon production was linked through the effect of climate on the growth of sockeye salmon (Oncorhynchus nerka) at sea. Smolt length and juvenile, immature, and maturing growth rates were estimated from increments on scales of adult sockeye salmon that returned to the Karluk River and Lake system on Kodiak Island, Alaska, over 77 years, 1924–2000. Survival was higher during the warm climate regimes and lower during the cool regime. Growth was not correlated with survival, as estimated from the residuals of the Ricker stock-recruitment model. Juvenile growth was correlated with an atmospheric forcing index and immature growth was correlated with the amount of coastal precipitation, but the magnitude of winter and spring coastal downwelling in the Gulf of Alaska and the Pacific Northwest atmospheric patterns that influence the directional bifurcation of the Pacific Current were not related to the growth of Karluk sockeye salmon. However, indices of sea surface temperature, coastal precipitation, and atmospheric circulation in the eastern North Pacific were correlated with the survival of Karluk sockeye salmon. Winter and spring precipitation and atmospheric circulation are possible processes linking survival to climate variation in Karluk sockeye salmon.

Implications of using On-Farm Flood Flow Capture to recharge groundwater and mitigate flood risks along the Kings River, CA

Two large hydrologic issues face the Kings Basin, severe and chronic overdraft of about 0.16M ac-ft annually, and flood risks along the Kings River and the downstream San Joaquin River. Since 1983, these floods have caused over $1B in damage in today’s dollars. Capturing flood flows of sufficient volume could help address these two pressing issues which are relevant to many regions of the Central Valley and will only be exacerbated with climate change. However, the Kings River has high variability associated with flow magnitudes which suggests that standard engineering approaches and acquisition of sufficient acreage through purchase and easements to capture and recharge flood waters would not be cost effective. An alternative approach investigated in this study, termed On-Farm Flood Flow Capture, involved leveraging large areas of private farmland to capture flood flows for both direct and in lieu recharge. This study investigated the technical and logistical feasibility of best management practices (BMPs) associated with On-Farm Flood Flow Capture. The investigation was conducted near Helm, CA, about 20 miles west of Fresno, CA. The experimental design identified a coordinated plan to determine infiltration rates for different soil series and different crops; develop a water budget for water applied throughout the program and estimate direct and in lieu recharge; provide a preliminary assessment of potential water quality impacts; assess logistical issues associated with implementation; and provide an economic summary of the program. At check locations, we measured average infiltration rates of 4.2 in/d for all fields and noted that infiltration rates decreased asymptotically over time to about 2 – 2.5 in/d. Rates did not differ significantly between the different crops and soils tested, but were found to be about an order of magnitude higher in one field. At a 2.5 in/d infiltration rate, 100 acres are required to infiltrate 10 CFS of captured flood flows. Water quality of applied flood flows from the Kings River had concentrations of COC (constituents of concern; i.e. nitrate, electrical conductivity or EC, phosphate, ammonium, total dissolved solids or TDS) one order of magnitude or more lower than for pumped groundwater at Terranova Ranch and similarly for a broader survey of regional groundwater. Applied flood flows flushed the root zone and upper vadose zone of nitrate and salts, leading to much lower EC and nitrate concentrations to a depth of 8 feet when compared to fields in which more limited flood flows were applied or for which drip irrigation with groundwater was the sole water source. In demonstrating this technology on the farm, approximately 3,100 ac-ft was diverted, primarily from April through mid-July, with about 70% towards in lieu and 30% towards direct recharge. Substantial flood flow volumes were applied to alfalfa, wine grapes and pistachio fields. A subset of those fields, primarily wine grapes and pistachios, were used primarily to demonstrate direct recharge. For those fields about 50 – 75% of water applied was calculated going to direct recharge. Data from the check studies suggests more flood flows could have been applied and infiltrated, effectively driving up the amount of water towards direct recharge. Costs to capture flood flows for in lieu and direct recharge for this project were low compared to recharge costs for other nearby systems and in comparison to irrigating with groundwater. Moreover, the potentially high flood capture capacity of this project suggests significant flood avoidance costs savings to downstream communities along the Kings and San Joaquin Rivers. Our analyses for Terranova Ranch suggest that allocating 25% or more flood flow water towards in lieu recharge and the rest toward direct recharge will result in an economically sustainable recharge approach paid through savings from reduced groundwater pumping. Two important issues need further consideration. First, these practices are likely to leach legacy salts and nitrates from the unsaturated zone into groundwater. We develop a conceptual model of EC movement through the unsaturated zone and estimated through mass balance calculations that approximately 10 kilograms per square meter of salts will be flushed into the groundwater through displacing 12 cubic meters per square meter of unsaturated zone pore water. This flux would increase groundwater salinity but an equivalent amount of water added subsequently is predicted as needed to return to current groundwater salinity levels. All subsequent flood flow capture and recharge is expected to further decrease groundwater salinity levels. Second, the project identified important farm-scale logistical issues including irrigator training; developing cropping plans to integrate farming and recharge activities; upgrading conveyance; and quantifying results. Regional logistical issues also exist related to conveyance, integration with agricultural management, economics, required acreage and Operation and Maintenance (O&M).

Evaluation and application of microsatellites for population identification of Fraser River chinook salmon (Oncorhynchus tshawytscha)

Variation at 13 microsatellite loci was previously surveyed in approximately 7400 chinook salmon (Oncorhynchus tshawytscha) sampled from 50 localities in the Fraser River drainage in southern British Columbia. Evaluation of the utility of the microsatellite variation for population-specific stock identification applications indicated that the accuracy of the stock composition estimates generally improved with an increasing number of loci used in the estimation procedure, but an increase in accuracy was generally marginal after eight loci were used. With 10–14 populations in a simulated fishery sample, the mean error in population-specific estimated stock composition with a 50-popula-tion baseline was <1.4%. Identification of individuals to specific populations was highest for lower Fraser River and lower and North Thompson River populations; an average of 70% of the individual fish were correctly assigned to specific populations. The average error of the estimated percentage for the seven populations present in a coded-wire tag sample was 2% per population. Estimation of stock composition in the lower river commercial net fishery prior to June is of key local fishery management interest. Chinook salmon from the Chilcotin River and Nicola River drainages were important contributors to the early commercial fishery in the lower river because they comprised approximately 50% of the samples from the net fishery prior to mid April.

Studies on blackening of canned prawns. Pt. 1. Influence of copper and iron on product blackening

A linear relationship was observed between the copper content and intensity of blackening in commercially canned prawn meat. Average copper and iron contents of non-blackened canned prawn meat were 9.6 and 32.5 ppm on dry weight basis respectively. In the blackened product copper content ranged from 15.8 to 63.9 ppm and iron content between 43.7 and 71.45 ppm depending on the intensity of blackening. But incorporation of copper in the above range to experimental cans produced blackening while iron up to 250 ppm did not impart any blackening under standard conditions of canning.

The residue measurment of pesticide in water and biota of the Sorkhrood River in the south of the Caspian Sea

Pesticide residue is considered as a crucial factor in the fresh water ecosystem pollution of north Iran (Esp. Gillan and Mazandaran provinces). It is also regarded dangerous to the health of human being and animals. In this research , pesticide residue in 3 species of fish was gauged in stations of the Haraz river , the Sorkhrood river , and the Caspian sea. The sampeles were taken during 27 mounths (started in April 97 and ended to June 99). The maximum level of pesticide in water was found in July 98 for lindine at 12.4 ppb. The level of chlorinated pesticide residue recorded at 63 ppb in April 98. The level of. organophosphorus pesticides residue in water was at 1.75 ppb in Aug 97. The toxicity of the mentioned pesticide and bioaccomulation were taken into account in conducting this researched.

Otolith characteristics and age determination of an endemic Ptychobarbus dipogon (Regan, 1905) (Cyprinidae: Schizothoracinae) in the Yarlung Tsangpo River, Tibet

We describe the microincrements, checks and annuli in the lapilli of the schizothoracine Ptychobarbus dipogon, an endemic species of the Tibetan plateau. We collected samples in the Yarlung Tsangpo River and its tributaries on a monthly basis (from April 2004 to August 2006). We describe the shape features of the three pairs of otoliths and document the full trajectory of lapillus development. We found that five to seven checks were clearly visible in the opaque zone of the first annulus. The pattern of 21-23 daily growth increments within each check might be explained as a lunar-induced deposition. We counted between 137 and 154 increments within the first annulus. Annuli appeared as a sequence of gradually declining increment widths, whereas false rings were characterized by abrupt checks. Our oldest estimates were 23(+)years for males and 44(+) for females. The time of annulus completion was clearly between March and April each year using monthly marginal increments analysis. We consider the factors responsible for daily increment formation as an endogenous circadian rhythm. Environmental information, such as strong sunlight and cold water temperatures in the Tibetan Plateau, could reinforce the endogenous daily cycle. Our results provided important data addressing the ecology and population dynamics of P. dipogon.

Age and growth of Schizopygopsis younghusbandi younghusbandi in the Yarlung Zangbo River in Tibet, China

Schizopygopsis younghusbandi younghusbandi is an endemic species whose distribution is restricted to the middle reaches of the Yarlung Zangbo River, being one of the most important commercial fishes in this area. Age and growth of 606 specimens captured between October 2002 and April 2005 were studied. The range in standard length (L) was 65.7-387.3 mm and total weight (W) was 3.3-772.0 g. The relationship between L and W was W=0.000909L(2.2493) for males and W=0.000259L(2.4781) for females. Age, determined from anal scales and lapillus otoliths, ranged from 3 to 18 years. The parameters of von Bertalanffy growth functions, estimated by back-calculated length, were L-infinity = 442.7mm L, k=0.0738 year(-1) and t(0)=-1.4 year for males, and L-infinity = 471.4mm L, k=0.0789 year(-1) and t(0)=0.2 year for females. Males and females exhibited statistically significant differences in growth. chi(2)-test indicated that von Bertalanffy growth functions could well describe the growth of S. y. younghusbandi. The longevities were 39.2 and 38.2 years for males and females, respectively. Growth inflexion points were 10.2 and 12.0 years for males and females, respectively, but 84.8% of the captures were at the smaller ages. So conservation and management schemes for this population should be considered urgently. In addition, we found that populations from the upstream of the Lhasa River, the downstream of the Lhasa River and the middle reaches of the Yarlung Zangbo River showed statistically significant differences in growth patterns.

Post-impoundment biomass and composition of phytoplankton in the Yangtze River

Damming, and thus alteration of stream flow, promotes higher phytoplankton populations and encourages algal blooms (density > 10(6) cells L-1) in the Three Gorges Reservoir (TGR). Phytoplankton composition and biomass were studied in the Yangtze River from March 2004 to May 2005. 107 taxa were identified. Diatoms were the dominant group, followed by Chlorophyta and Cyanobacteria. In the Yangtze River, algal abundance varied from 3.13 x 10(3) to 3.83 x 10(6) cells L-1, and algal biomass was in the range of 0.06 to 659 mg C m(-3). Levels of nitrogen, phosphorus and silica did not show consistent longitudinal changes along the river and were not correlated with phytoplankton parameters. Phytoplankton abundance was negatively correlated with main channel discharge (Spearman r = -1.000, P < 0.01). Phytoplankton abundance and biomass in the Yangtze River are mainly determined by the hydrological conditions rather than by nutrient concentrations.

Sediment sources and the flood record from Wanghu lake, in the middle reaches of the Yangtze River

A sediment core was collected from the centre of Wanghu Lake, in the Middle Reaches of the Yangtze River. The recent part of the core was dated using a combination of Pb-210 and spheroidal carbonaceous particle (SCP) techniques. Extrapolating this chronology dated the laminated section of the core, between 723 and 881 mm, to the first half of the 18th century and this section was selected for detailed study. The thicknesses of the laminae were measured using reflecting and polarizing microscopes whilst geochemistry was determined by an electron probe. The thickness of the dark layers was found to be positively correlated with titanium concentrations, and negatively correlated with aluminium and potassium concentrations. The thickness of the light layers was found to be negatively correlated with the concentrations of titanium. It is concluded that the dark layers were deposited from the Fushui River, a tributary of the Yangtze River, under periods of normal flow whilst the light Layers were mainly deposited from the Yangtze River itself during flood periods. Documentary evidence for floods occurring in the take catchment corresponded with thick laminations of high titanium concentration. Further, two of the three thickest, light laminations with low titanium concentrations were found to be synchronous with recorded flood dates of the main Yangtze River in its Middle Reaches, but one was synchronous with a local drought. These data suggest that the Lake sediment provides an archive of the relative water levels of the Yangtze and Wanghu including floods of both the main Yangtze River and the local hydrological regime. (c) 2006 Elsevier B.V. All rights reserved.

DNA polymorphism of the plankton community and its relationships to species composition in the Three Gorges Reservoir Region of the Yangtze River

We examined DNA polymorphism of the plankton community in the Three Gorges Reservoir Region of Yangtze River and studied its relationships to species composition. Samples of the plankton community were collected from nine sampling sites and analyzed by random amplified polymorphic DNA (RAPD). Nine of 60 screened primers generated a total of 88 observable 180 to 1400 by bands, all of which were polymorphic. Cluster analysis of the resulting binary format from DNA banding patterns grouped the target communities into three clusters. The topology of the constructed diagram from species composition data was generally similar to that based on RAPD markers.