Thesis (M.S.)--West Virginia University, 1998. / Title from document title page. On t.p. "1̳" is subscript. Document formatted into pages; contains xi, 49 p. : ill. Includes abstract. Includes bibliographical references (p. 46-48).

Thesis (Ph. D.)--Ohio State University, 2005. / Title from first page of PDF file. Document formatted into pages; contains xxiii, 184 p.; also includes graphics (some col.). Includes bibliographical references (p. 169-184). Available online via OhioLINK's ETD Center

No description available.

C-type cytochromes are ubiquitous proteins with crucial functions in organisms, which include electron transfer and apoptotic signalling. In eukaryotic organisms, mitochondrial cytochrome c is located in the intermembrane space, and it is a component of the electron transport chain; it is responsible for transferring electrons from Complex III to Complex IV. The regulated release of cytochrome c from mitochondria results in the activation of a signal transduction pathway leading to controlled cell death, or apoptosis. In mitochondrial c-type cytochromes, the heme is bound to both cysteines of a CXXCH motif located near the N-terminus. The covalent heme attachment in c-type cytochromes, the final step in its biosynthesis, is achieved by different cytochrome c biogenesis systems in different organisms. Out of these systems, System III, found in many eukaryotes, has a single component - holocytochrome c synthase (HCCS) which is the enzyme responsible for the catalysis of heme binding to cytochrome c. HCCS recognises apocytochrome c as a substrate upon the import of the apocytochrome from the cytosolic space to the mitochondrial intermembrane space. The requirements of amino acid sequence for HCCS recognition had remained an intriguing question, despite the relatively long period since the discovery of the enzyme. Thus, HCCS in System III and its substrate recognition is the subject of this thesis. This thesis describes the experiments showing that the N-terminal region of the mitochondrial cytochrome c protein is important for substrate recognition, as well as further characterisation of this sequence by mutagenesis. Out of several highly conserved residues in the N-terminus, a phenylalanine residue in the N-terminus is identified to be critical for heme attachment by HCCS. The role of this phenylalanine residue in the interaction between the two proteins was probed by substituting it with a range of residues. Furthermore, the importance of the spacing between the key phenylalanine residue and the CXXCH motif was investigated. A single-cysteine variant of the mitochondrial cytochrome c with a single bond to the heme is produced by HCCS, but heme attachment only occurs if histidine is present as an axial ligand to the heme iron. Replacement of the histidine with other potential iron-ligating residues abolished heme attachment. These results bring insight into the critical features in amino acid sequence of cytochrome c for the substrate recognition specificity of HCCS. Sequence analysis on the N-terminal region of mitochondrial cytochromes c in a variety of organisms reveals evolutionary implications for cytochrome c biogenesis systems. It also attempts to explain the reason for negative results in previous chapters for the analysis of the N-terminal region of cytochrome c. An improved method for human HCCS production is also described in this thesis, for the exploitation of purification and characterisation in future studies of HCCS.

The crystal structure of yeast (Saccharomyces cerevisiae) iso-1- cytochrome c has been determined through molecular replacement techniques, and refined against X-ray diffraction data in the resolution range 6.0-1.23 Å to a crystallographic R-factor of 0.192. The yeast iso-1-cytochrome c molecule has the typical cytochrome c fold, with the polypeptide chain organized into five α-helices and a series of loops which serve to enclose almost completely the heme prosthetic group within a hydrophobic pocket Comparison of the structures of yeast iso-1-, tuna and rice cytochromes c shows that the polypeptide backbone fold, intramolecular hydrogen bonding, conformation of side chains and particularly packing within the heme crevice of protein groups against the heme moiety are very similar in the three proteins. Significant structural differences among the three cytochromes c can be explained by differences in amino acid sequence. X-ray crystallographic techniques have also been used to study the effect of single-site amino acid substitutions at Phe82 and at Arg38 in iso-1-cytochrome c. The structures of the various variant iso-1-cytochromes c have been determined at nominal resolutions in the range 2.8 to 1.76 Å. Conspicuous structural perturbations in the neighborhood of the substituted side chain are evident in all of the variant proteins. In wild-type iso-1-cytochrome c, the phenyl ring of Phe82 is positioned adjacent and approximately parallel to the heme group, and occupies a non-polar cavity within the heme crevice. In the Ser82 variant, a channel extending from the surface of the molecule down into the heme crevice is created. In the Gly82 variant, the polypeptide backbone has refolded into the space formerly occupied by the phenyl ring of Phe82. Steric conflicts prevent both the phenolic ring of Tyr82 and the side chain of Ile82 from being completely accommodated within the pocket normally occupied by a phenyl ring. Substitution of alanine at position 38 causes a slight reorganization of the hydrogen bonding network in which Arg38 normally participates, and also exposes to external solvent a normally buried propionic acid group of the heme. The altered functional properties of the position 82 variant proteins have been interpreted with respect to the observed structural perturbations. The drop in reduction potential, most notably for the Ser82 and Gly82 variants, can be explained by the elevated heme environment polarity arising from the increased access of solvent or polar protein groups to the heme pocket The reduced stability of the heme crevice, as indicated by lowered pKa's for alkaline isomerization, is likely due to the disruption of stabilizing packing forces formed by the Phe82 phenyl ring within its hydrophobic cavity. The lowered activity, in comparison to the wild-type protein and the Tyr82 variant, for electron transfer with Zn+-cytochrome c peroxidase is attributed to the loss of an aromatic group positioned adjacent to the heme group. The altered surface topography of the variant proteins (particularly the Gly82, Tyr82 and Ile82 variants) may further hinder productive complex formation between cytochrome c and its redox partners. These results suggest that the invariant Phe82 contributes in at least three ways to the proper functioning of cytochrome c. It has an important structural role in maintaining the integrity of the heme crevice and in establishing the appropriate heme environment The phenyl ring of Phe82 may also be required for efficient movement of an electron to and from the heme of cytochrome c. Finally, Phe82 may have a role in forming intermolecular interactions with enzymic redox partners of cytochrome c. / Medicine, Faculty of / Biochemistry and Molecular Biology, Department of / Graduate

Yes / The identification and quantification of functional cytochromes P450 (CYPs) in biological samples is proving important for robust analyses of drug efficacy and metabolic disposition. In this study, a novel CYP activity-based probe was rationally designed and synthesised, demonstrating selective binding of CYP isoforms. The dependence of probe binding upon the presence of NADPH permits the selective detection of functionally active CYP. This allows the detection and analysis of these enzymes using biochemical and proteomic methodologies and approaches. / Engineering and Physical Sciences Research Council (EPSRC) and Yorkshire Cancer Research

Mémoire numérisé par la Direction des bibliothèques de l'Université de Montréal.

The lens is responsible for focusing light into the retina. It accomplishes this through its maturation from an epithelial cell into a fiber cell. A large amount of research has been done on cellular differentiation. Nevertheless, we still lack knowledge on many different aspects of differentiation, including a complete theory on the mechanism behind differentiation. Due to the lens’ unique structure and cell types, this is an ideal model for studying differentiation. Our research has shown that αB crystallin, a small heat shock protein, is able to modulate cytochrome C levels and protect the mitochondria under oxidative stress. Also, cytochrome C release is often followed by caspase 3 activation. In addition, research has shown that low levels of caspase 3 activation is essential in driving differentiation. My work examined if αB crystallin could modulate cytochrome C to lower caspase 3 levels to allow for differentiation rather than apoptosis. / Includes bibliography. / Thesis (M.S.)--Florida Atlantic University, 2019. / FAU Electronic Theses and Dissertations Collection

This thesis describes a study of the substrate selectivity of active site mutants of the monooxygenase cytochrome P450_cam. A range of mutants was constructed which replaced the phenolic side-chain at the Tyr-96 position by various hydrophobic amino acid residues. These 'hydrophobic mutants' were then combined with other mutations around the active site (Val-247, Phe-87, Ile-395 and Phe-193) which altered the space available at different positions in the active site. These mutants were then tested with an in vitro reconstituted P450_cam system with a range of substrates related to diphenylmethane and phenylcylcohexane. All of these large compounds were poor substrates for the wild-type enzyme. It was found that it was necessary to increase both the space available in the active site and the active site hydrophobicity to achieve substrate turnover. The substrates were oxidised preferentially on the aliphatic cyclohexyl ring over the more constrained phenyl ring suggesting that the active site is predisposed to binding the cyclohexyl ring close to the haem. Hydroxylation using the in vitro reconstituted P450_cam system is limited by catalyst lifetime and the need for the expensive cofactor NADH. For P450_cam hydroxylation to become a viable synthetic method it is necessary to find ways to bypass the use of NADH. For this reason various self-sufficient P450_cam system were constructed and expressed in E. coli. The best of these, despite limited protein expression, was found to turnover camphor with the wild-type P450_cam enzyme and other substrates with the Y96A mutant. The in vivo catalytic system was then used to screen many P450_cam mutants for the oxidation of natural products, monoterpenes and sesquiterpenes (e.g. limonene, pinene and valencene). Most of the target substrates are not oxidised by the wild-type enzyme but all are hydroxylated by some if not all of the P450_cam mutants with different degrees of selectivity. Some of the products identified so far are important compounds in the field of flavour and fragrance chemistry (e.g. verbenol and nookatone).

Thesis (M.S.)--Brigham Young University. Dept. of Biology, 2007. / Includes bibliographical references.