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.

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>

No description available.

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.

Cu+-ATPases are transmembrane enzymes that couple the efflux of cytoplasmic Cu+ to the hydrolysis of ATP. It is well established that Cu+-ATPases control cytoplasmic Cu+ levels. However, bacterial genomes, particularly those of symbiotic/pathogenic organisms, contain multiple copies of genes encoding Cu+-ATPases, challenging the idea of a singular role for these enzymes. Our lab has demonstrated that one of the two Cu+-ATPases in Pseudomonas aeruginosa, a FixI-type ATPase, has an alternative role, most likely Cu+ loading of cytochrome c oxidase (Cox). To further study alternative roles of Cu+-ATPases, we study the symbiont Sinorhizobium meliloti. Rhizobia are soil-dwelling bacteria that interact with legumes, forming plant root nodules that actively fix N2. The S. meliloti genome contains five Cu+-ATPases, two of which are FixI-type. Both of these enzymes, termed FixI1 and FixI2, are downstream of Cox operons. We hypothesized that the presence of multiple FixI-type ATPases was not an example of redundancy, but rather is an evolutionary adaptation that allows rhizobia to survive under the wide variety of adverse conditions faced during early infection and establishment of symbiosis. Towards this goal, this work focused on examining the effects of mutation of each ATPase on both free-living bacteria and on the ability of rhizobia to establish an effective symbiosis with its host legume. Each of these mutants presents a different phenotype at varying points of the nodulation process, and only the fixI2 mutation produces a respiratory-deficient phenotype during aerobic growth. These results are consistent with our hypothesis that the two proteins have non-redundant physiological functions. Understanding the factors that contribute to an effective symbiosis is beneficial, since N2 fixation in legumes is important to both agriculture and industry.

Many fish species respond to cold temperatures by inducing mitochondrial biogenesis, reflected in an increase in the activity of the mitochondrial enzyme cytochrome c oxidase (COX). COX is composed of 13 subunits, 3 encoded by mtDNA and 10 encoded by nuclear genes. I used thermal acclimation/winter acclimatization to explore how fish muscle controls the synthesis of COX. In this study, I used real-time PCR to measure mRNA levels for the 10 nuclear-encoded COX genes and several transcriptional regulators. I compared the thermal response of two cyprinid species, the tropical zebrafish (Danio rerio, acclimated to 11 and 30°C) and the temperate redbelly dace (Phoxinus eos, winter and summer acclimatized). I hypothesized that (i) there would be an increase in COX activity in the cold- versus warm-acclimated fish and (ii) changes in COX activity would be paralleled in the transcript levels of the nuclear-encoded COX subunits as well as the master-regulators and transcription factors of mitochondrial biogenesis. Zebrafish COX activity did not change in the cold but the transcript levels of some subunits decreased up to 70%. Redbelly dace COX activity was 2.9-fold higher in winter fish and though nuclear-encoded subunits had higher transcript levels the increases did not parallel enzyme activity, ranging from 1.7- to 21-fold higher in winter. There also did not appear to be parallel patterns in mRNA for the transcriptional regulators. In zebrafish, when COX activity did not change, there was no significant change in PGC-1α mRNA. In redbelly dace, when COX activity was 2.9-fold higher, PGC-1α mRNA was 6.3-fold higher. These observations suggest that coordination of COX subunit expression is imperfect, implying that subsets of these genes are more important in determining the COX activity. I assert that those genes that are most likely the candidates for regulating COX activity are COX4 and COX5A as they are the first regulatory subunits incorporated into the holoenzyme. Though arguments can also be made for COX5B, 6A and 7B based on the parallels between changes in enzyme activity and transcript abundance as well as the position in which they are assembled into the enzyme complex. / Thesis (Master, Biology) -- Queen's University, 2010-08-10 11:35:35.352

Thesis (M.Sc.) --University of Adelaide, 1946. / Typewritten copy.

No description available.

Cytochrome c oxidase (COX) is complex IV of the electron transport system, and catalyzes the reduction of molecular oxygen to water. It possesses ten nuclear-encoded subunits, the largest of which is COX4. Bayesian analysis suggests the isoform pair for this subunit arose early in vertebrate evolution, and tissue distribution of the COX4 paralogs is similar in mammals and teleosts: COX4-1 is ubiquitously transcribed while COX4-2 is present in large amounts only in brain and respiratory tissue. This subunit is of interest due to its apparent sensitivity to oxygen. During hypoxia, transcription switches from COX4-1 to COX4-2 in some mammalian tissues. However, questions remain about the regulation of this response as well as its pervasiveness across vertebrates. I investigated these uncertainties by measuring the transcriptional response of the COX4 paralogs to hypoxia in a variety of vertebrate models, and assessing the hypoxic induction of putative oxygen-responsive elements (HRE1, HRE2, and ORE) from candidate vertebrate species in a transfection experiment. I also examined the conservation of key elements of the COX4-2 gene and polypeptide in vertebrates. It was found that the hypoxia-responsiveness of COX4-2 may not be vital to the cellular response to hypoxia. COX4-1 transcripts remained in excess during hypoxia in all of the vertebrate models used with the exception of western painted turtle (Chrysemys picta), where COX4-2 transcripts remained in excess during control and hypoxic treatments. Only the HRE2 element from human COX4-2 was activated with hypoxic exposure, yet this along with the other features of the gene and polypeptide were not well conserved across mammals, and nearly absent outside of this lineage. These results provide evidence that COX4-2 may respond to hypoxia in only select few mammalian tissues, or that the function of this gene is not related to the cellular hypoxic response. / Thesis (Master, Biology) -- Queen's University, 2012-11-25 20:51:59.419

Copper is an essential cofactor of two mitochondrial enzymes: cytochrome c oxidase (COX) and the mitochondrial localized fraction of Cu-Zn superoxide dismutase (Sod1p). Copper incorporation into these enzymes is facilitated by a growing number of metallochaperone proteins. Here we describe two novel copper chaperones of COX, Cmc1 and Cmc2. In Saccharomyces cerevisiae, both Cmc1 and Cmc2 localize to the mitochondrial inner membrane facing the intermembrane space. Cmc1 and Cmc2 are essential for full expression of COX and cellular respiration, contain a twin Cx9C domain, and are conserved from yeast to humans. Additionally, the presence or absence of these proteins not only determines full assembly of functional COX but also affects metallation of Sod1 suggesting these proteins might play a role on co-modulation of copper transfer to COX and Sod1. CMC1 overexpression does not rescue the respiratory defect of cmc2 mutants or vise versa. However, Cmc2 physically interacts with Cmc1 and the absence of Cmc2 induces a 5-fold increase in Cmc1 accumulation in the mitochondrial membranes. We conclude that Cmc1 and Cmc2 have cooperative but non-overlapping functions in cytochrome c oxidase biogenesis.