Includes bibliography.

This thesis reports the synthesis and copper coordination chemistry of covalently-linked aryl-imidazole derivatives designed as models for the crosslinked imidazole-phenol sidechains of the His-Tyr cofactor in the CcO. Three new imidazole- (HL1 - HL3) and three new indole- (HL4 - H2L6) containing tripodal ligands were synthesised. The conjugate addition of an imidazole to activated quinone derivatives was developed as a new route to organic models for the Tyr His cofactor. Two monodentate imidazole-aryl, Im-hq(OH)2 and Im-ArOH, and an imidazole-quinone, Im bq were obtained using this route. The X-ray crystal structure of Im-hq(OH)2.EtOH was determined. The route was also used to give new chelating ligands, H2L10 and HL12, containing a cross-linked imidazole-phenol surrogate for the Tyr244-His240 cofactor. Copper complexes of Im-hq(OH)2, Im-bq, Im-ArOH, H2L10-HL12, and HL1-H2L6 were prepared, and the X-ray crystal structures of [Cu(terpy)(Im-bq)][BF4]2 and five other copper complexes were determined. The physiochemical properties of the copper complexes were characterized by FT-IR, UV-Vis-NIR, EPR and (spectro)electrochemical studies. Key results include: the oxidation of Im-ArO- anion affords the semiquinone radical, Im-sq(4OH)(1O??????), in a hydrous solvent. However, the oxidations of neutral Im-ArOH and [Cu(tpa)(Im-ArOH)]2+ produce the corresponding phenoxy radical species that rapidly and reversibly dimerise to give quinol cyclohexadienone, QCHD, dimers. Significantly [Cu(tpa)(Im-sq(4OH)(1O??????))]2+ was EPR silent, perhaps due to antiferromagnetic coupling between the Cu(II) (S=1/2) and semiquinonyl radical (S=1/2) centres. Deprotonation of the hydroquinone in [Cu(tpa)(Im-hq(OH)2]2+ produces the hydroquinone dianion which reduces the Cu(II) centre. The semiquinone radical is coordinatively labile and dissociates from the Cu(I) centre. The biological implications of these results are mentioned.

Richardson ground squirrels (gophers) are pests on the prairies that cause considerable agricultural and ecological damage. Traditional control methods such as the rodenticides strychnine, zinc phosphide, and anticoagulants, have proven ineffective in reducing gopher densities. In additional, current gopher control methods have the significant potential to cause primary and secondary toxicity to non-target animals. Thus, alternative methods for toxicological control of gophers are needed to mitigate these concerns. Present studies focused on the cytochrome P450 (CYP450) enzyme system responsible for xenobiotic detoxification in gophers. In vitro hepatic microsomal systems and HPLC analysis were used to elucidate general metabolic characteristics of major gopher xenobiotic metabolizing pathways. We found that the content and activity of individual components of the CYP450 system including CYP450, cytochrome b5, and NADPH-cytochrome P450 reductase in liver microsomal preparations were higher in gophers exposed to toxins used to control their population than in naïve (unexposed) gophers. When in vitro CYP450 mediated activities for five substrates [coumarin and aniline aromatic hydroxylation, 7-methoxycouamrin O-demethylation, and N-methylaniline, and N,N-dimethylaniline N-demethylation] were measured, naïve gophers were identified to have higher specific activity but similar whole body activity compared to the exposed gophers. Furthermore, there was a clearly identifiable sub-population of poor metabolizers showing considerably lower CYP450 activity within the gopher samples studied. Clotrimazole was found to be a potent inhibitor of several substrates of CYP450 enzyme-mediated reactions, which included aniline aromatic hydroxylation, N-methylaniline and N,N-dimethylaniline N-demethylation, and 7-methoxycoumarin O-demethylation. The cytotoxicity of above compounds was tested using freshly isolated gopher hepatocytes. The results showed that each compound caused considerable cytotoxicity to gopher hepatocytes. Addition of clotrimazole to the freshly isolated hepatocyte suspension increased the cytotoxicity of all tested compounds. <p>In conclusion, gophers may develop resistance to current chemical control methods through the enhancement of CYP450 system content, which can compensate the loss of enzyme activity. Furthermore, clotrimazole is a potent cytochrome P450 inhibitor, which increases the cytotoxicity caused by given compounds in gopher livers. The concept of using CYP450 enzyme inhibitor in combination with another chemical whose elimination depends on CYP450 metabolism to improve current gopher control method has practical importance.

Proton transfer is one of the most common reactions in biological systems. During energy conversion inside a cell, proton transfer is crucial to maintain an electrochemical proton gradient across the cell membrane. This gradient is in turn used to e.g. produce ATP, the energy currency of the cell. One of the key components of the build-up of this gradient is cytochrome c oxidase. This membrane-bound enzyme catalyzes the reduction of molecular oxygen to water, using protons and electrons, and in the process protons are pumped across the membrane. All protons used during oxygen reduction and those that are pumped, are transferred via hydrophilic pathways inside the hydrophobic interior of the enzyme. One of these pathways, called the D pathway, is used to transfer protons both to the catalytic site and towards a pump site. It is yet not fully understood how these proton-transfer reactions are timed, coupled and controlled.   This thesis is focused on studies of proton-transfer reactions through the D pathway in variants of cytochrome c oxidase that lack the ability to pump protons. The results suggest that changes in pKa values of key residues, as well as structural changes inside the pathway, can explain the non-pumping phenotypes. The results have led us to propose that an internal proton shuttle (Glu286I) can adopt two different conformations that are in equilibrium with each other, and that this equilibrium is altered in non-pumping variants of cytochrome c oxidase. We also observed that proton transfer through the D pathway could occur with the same rate as in the wild-type enzyme even when one of the key residues (Asp132I) is absent. This result contradicts previous assumptions that acidic residues must be present at an orifice of proton pathways. We therefore suggest that this specific residue could have an additional role, e.g. as a selectivity filter that excludes all ions except protons from entering the pathway. / <p>At the time of doctoral defence the following papers were unpublished and had a status as follows: Paper 2: Accepted; Paper 3: Manuscript</p>

Richardson ground squirrels (gophers) are pests on the prairies that cause considerable agricultural and ecological damage. Traditional control methods such as the rodenticides strychnine, zinc phosphide, and anticoagulants, have proven ineffective in reducing gopher densities. In additional, current gopher control methods have the significant potential to cause primary and secondary toxicity to non-target animals. Thus, alternative methods for toxicological control of gophers are needed to mitigate these concerns. Present studies focused on the cytochrome P450 (CYP450) enzyme system responsible for xenobiotic detoxification in gophers. In vitro hepatic microsomal systems and HPLC analysis were used to elucidate general metabolic characteristics of major gopher xenobiotic metabolizing pathways. We found that the content and activity of individual components of the CYP450 system including CYP450, cytochrome b5, and NADPH-cytochrome P450 reductase in liver microsomal preparations were higher in gophers exposed to toxins used to control their population than in naïve (unexposed) gophers. When in vitro CYP450 mediated activities for five substrates [coumarin and aniline aromatic hydroxylation, 7-methoxycouamrin O-demethylation, and N-methylaniline, and N,N-dimethylaniline N-demethylation] were measured, naïve gophers were identified to have higher specific activity but similar whole body activity compared to the exposed gophers. Furthermore, there was a clearly identifiable sub-population of poor metabolizers showing considerably lower CYP450 activity within the gopher samples studied. Clotrimazole was found to be a potent inhibitor of several substrates of CYP450 enzyme-mediated reactions, which included aniline aromatic hydroxylation, N-methylaniline and N,N-dimethylaniline N-demethylation, and 7-methoxycoumarin O-demethylation. The cytotoxicity of above compounds was tested using freshly isolated gopher hepatocytes. The results showed that each compound caused considerable cytotoxicity to gopher hepatocytes. Addition of clotrimazole to the freshly isolated hepatocyte suspension increased the cytotoxicity of all tested compounds. <p>In conclusion, gophers may develop resistance to current chemical control methods through the enhancement of CYP450 system content, which can compensate the loss of enzyme activity. Furthermore, clotrimazole is a potent cytochrome P450 inhibitor, which increases the cytotoxicity caused by given compounds in gopher livers. The concept of using CYP450 enzyme inhibitor in combination with another chemical whose elimination depends on CYP450 metabolism to improve current gopher control method has practical importance.

Thèse de doctorat : Chimie : Strasbourg : 2009. / Titre provenant de l'écran-titre. Bibliogr. 7 p.

Thesis (Ph. D.)--Washington State University, December 2009. / Title from PDF title page (viewed on Dec. 11, 2009). "School of Molecular Biosciences." Includes bibliographical references.

Thesis (M. Phil.)--University of Hong Kong, 2001. / Includes bibliographical references.

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