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.

Biological electron transfer in copper proteins

Nature has used a variety of protein systems to mediate electron transfer. In this thesis I examine aspects of the control of biological electron transfer by two copper proteins that act as natural electron carriers.

In the first study, I have made a mutation to one of the ligand residues in the azurin blue copper center, methionine 121 changed to a glutamic acid. Studies of intramolecular electron transfer rates from that mutated center to covalently attached ruthenium complexes indicate that the weak axial methionine ligand is important not only for tuning the reduction potential of the blue copper site but also for maintaining the low reorganization energy that is important for fast electron transfer at long distances.

In the second study, I begin to examine the reorganization energy of the purple copper center in the CuA domain of subunit II of cytochrome c oxidase. In this copper center, the unpaired electron is delocalized over the entire binuclear site. Because long-range electron transfer into and out of this center occurs over long distances with very small driving forces, the reorganization energy of the CuA center has been predicted to be extremely low. I describe a strategy for measuring this reorganization energy starting with the construction of a series of mutations introducing surface histidines. These histidines can then be labeled with a series of ruthenium compounds that differ primarily in their reduction potentials. The electron transfer rates to these ruthenium compounds can then be used to determine the reorganization energy of the CuA site.

An experimental study of the mobility of edge dislocations in pure copper single crystals

The velocity of selectively-introduced edge dislocations in 99.999 percent pure copper crystals has been measured as a function of stress at temperatures from 66°K to 373°K by means of a torsion technique. The range of resolved shear stress was 0 to 15 megadynes/ cm^2 for seven temperatures (66°K, 74°K, 83°K, 123°K, 173°K, 296°K, 296°K, 373°K.

Dislocation mobility is characterized by two distinct features; (a) relatively high velocity at low stress (maximum velocities of about 9000 em/sec were realized at low temperatures), and (b) increasing velocity with decreasing temperature at constant stress.

The relation between dislocation velocity and resolved shear stress is:

v = v_o(τ_r/τ_o)^n

where v is the dislocation velocity at resolved shear stress τ_r, v_o is a constant velocity chosen equal to 2000 cm/ sec, τ_o is the resolved shear stress required to maintain velocity v_o, and n is the mobility coefficient. The experimental results indicate that τ_o decreases from 16.3 x 10^6 to 3.3 x 10^6 dynes/cm^2 and n increases from about 0.9 to 1.1 as the temperature is lowered from 296°K to 66°K.

The experimental dislocation behavior is consistent with an interpretation on the basis of phonon drag. However, the complete temperature dependence of dislocation mobility could not be closely approximated by the predictions of one or a combination of mechanisms.

The distribution of native and introduced species of crayfish in Austria

This account concentrates on the six species of crayfish found in Austria, and the current state of knowledge on their distribution and laws affecting conservation. In general the occurrence and distribution of crayfish in Austria is poorly known, although information obtained by researchers and the general public, after careful checking, is increasing. Three native crayfish species occur in Austria: Austropotamobius torrentium which is relatively widespread, A. pallipes with a restricted distribution, and Astacus astacus which is widespread. Three species of non-native (alien) crayfish have been recorded from a total of 158 localities in Austria. They are Astacus leptodactylus from eastern Europe, and two Nearctic species: Pacifastacus leniusculus and Orconectes limosus. The introduction of alien species causes considerable problems as they act as vectors of crayfish plague and are able to outcompete native species by higher reproductive capacities.

An assessment of post-plague reintroduced native white-clawed crayfish, a Ustropotamobius pallipes, in the Sherston Avon and Tetbury Avon, Wiltshire

This article reports on the success of reintroducing native crayfish (Austropotamobius pallipes) in the Sherston and Tetbury Avon, following extinction of the population from crayfish plague. The authors describe and review the survey methods that were used and identify a survey technique that was found to be the most rapid and robust for monitoring crayfish populations. Such a survey technique could be adopted as a standard method.

The current distribution of signal and native crayfish in the Broadmead Brook, Wiltshire

Signal crayfish (Pacifastacus leniusculus) have existed in the upper reaches of Broadmead Brook in Wiltshire since 200 individuals were introduced at West Kington in 1981. The population has expanded upstream and downstream since this introduction, however, giving rise to concerns that it may potentially threaten the native crayfish population further downstream. Signal crayfish can act as a vector of crayfish plague - a disease caused by the fungus Aphanomyces astaci Schikora which results in almost complete mortality to the native, white-clawed crayfish Austropotamobius pallipes. The native crayfish in Broadmead Brook have not yet succumbed to crayfish plague and are currently free of the disease. However, as signal crayfish appear to out-compete the native species, the native population could still be under threat. In this article, we highlight the findings of previous crayfish surveys on Broadmead Brook and describe work undertaken in summer 2001 to map the current distribution of native and signal crayfish. Finally, options for controlling the spread of signal crayfish are discussed.

Expanding the Toolkit for Synthetic Biology: Frameworks for Native-like Non-natural Gene Circuits

Synthetic biology combines biological parts from different sources in order to engineer non-native, functional systems. While there is a lot of potential for synthetic biology to revolutionize processes, such as the production of pharmaceuticals, engineering synthetic systems has been challenging. It is oftentimes necessary to explore a large design space to balance the levels of interacting components in the circuit. There are also times where it is desirable to incorporate enzymes that have non-biological functions into a synthetic circuit. Tuning the levels of different components, however, is often restricted to a fixed operating point, and this makes synthetic systems sensitive to changes in the environment. Natural systems are able to respond dynamically to a changing environment by obtaining information relevant to the function of the circuit. This work addresses these problems by establishing frameworks and mechanisms that allow synthetic circuits to communicate with the environment, maintain fixed ratios between components, and potentially add new parts that are outside the realm of current biological function. These frameworks provide a way for synthetic circuits to behave more like natural circuits by enabling a dynamic response, and provide a systematic and rational way to search design space to an experimentally tractable size where likely solutions exist. We hope that the contributions described below will aid in allowing synthetic biology to realize its potential.

Absolute oscillator strengths for iron, copper, cadmium and gold measured by the atomic beam method

Absolute f-values for 7 transitions in the first spectra of 4 elements have been measured using the atomic beam absorption technique. The equivalent widths of the absorption lines are measured with a photoelectric scanner and the atomic beam density is determined by continuously weighing a part of it with a sensitive automatic microbalance. The complete theory is presented and corrections are calculated to cope with gas absorption by the deposit on the microbalance pan and atoms which do not stick to the pan. An additional correction for the failure of the assumption of effusive flow in the formation of the atomic beam at large densities has been measured experimentally.

The following f-values were measured:

Fe: f_λ3720 = 0.0430 ± 8%

Cu: f_λ3247 = 0.427 ± 4.5%, f_λ3274 = 0.206 ± 4.7%, f_λ2492 = 0.0037 ± 9%

Cd: f_λ3261 = 0.00190 ± 7%, f_λ2288 = 1.38 ± 12%

Au: f_λ2428 = 0.283 ± 5.3%

Comparison with other accurately measured f-values, where they exist, shows agreement within experimental errors.

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.

Metallothionein as an indicator of water quality: assessment of the bioavailability of cadmium, copper, mercury and zinc in aquatic animals at the cellular level

The study of metallothioneins (MTs) has greatly improved our understanding of body burdens, metal storage and detoxification in aquatic organisms subjected to contamination by the toxic heavy metals, Cd, Cu, Hg and Zn. These studies have shown that in certain organisms MT status can be used to assess impact of these metals at the cellular level and, whilst validation is currently limited to a few examples, this stress response may be linked to higher levels of organisation, thus indicating its potential for environmental quality assessment. Molluscs, such as Mytilus spp., and several commonly occurring teleost species, are the most promising of the indicator species tested. Natural variability of MT levels caused by the organism's size, condition, age, position in the sexual cycle, temperature and various stressors, can lead to difficulties in interpretation of field data as a definitive response-indicator of metal contamination unless a critical appraisal of these variables is available. From laboratory and field studies these data are almost complete for teleost fish. Whilst for molluscs much of this information is lacking, when suitable controls are utilised and MT measurements are combined with observations of metal partitioning, current studies indicate that they are nevertheless a powerful tool in the interpretation of impact, and may prove useful in water quality assessment.

Optical parameters and absorption of copper (II)-azo complexes thin films as optical recording media

Smooth thin films of three kinds of azo dyes of 2-(5'-tert-butyl-3'-azoxylisoxazole)-1, 3-diketones and their copper (II)-azo complexes were prepared by the spin-coating method. Absorption spectra of the thin films on a glass substrate in the 300-600 nm wavelength region were measured. Optical constants (complex refractive index N=n+ik) and thickness of the thin films prepared on single-crystal silicon substrate in the 300-600 nm wavelength region were investigated on rotating analyzer-polarizer type of scanning ellipsometer, and dielectric constants epsilon(epsilon=epsilon(1)+i epsilon(2)), absorption coefficients alpha as well as reflectance R of thin films were then calculated. In addition, one of the copper (II)-azo complex thin film prepared on glass substrate with an Ag reflective layer was also studied by atomic force microscopy (AFM) and static optical recording. AFM study shows that the copper (II)-azo complex thin film is very smooth and has a root mean square surface roughness of 1.89 nm. Static optical recording shows that the recording marks on the copper (II)-azo complex thin film are very clear and circular, and the size of the minimal recording marks can reach 200 nm. (c) 2004 Elsevier B.V. All rights reserved.