Due to the biological importance of heme and its implication in various disease states, uncovering how it is transported throughout the cell is of vital importance. Some of the strongest in vivo tools present in the literature are FRET-based sensors using a number of chromophores that are optimized and expanded from GFP. In order to elucidate the movement of heme throughout the cell, GFP FRET -based heme sensors were designed, expressed, and purified to be further characterized in vitro. This series of heme sensors were expressed in Saccharomyces cerevisiae to monitor the in vivo movement of heme. Different growth conditions were explored to monitor the effect of these changes to cytosolic heme availability. These heme sensors are now poised to address the movement of heme from the mitochondria to other targets in the cell under a variety of conditions. This will provide insight into heme trafficking pathways, as well as the role heme plays in neurodegenerative diseases and aging

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The majority of the work in this dissertation describes the characterization of the nitrophorins (NPs) by NMR spectroscopy.NP7 was studied and compared to NP2 and relevant mutants.NP7 is shown to have three additional amino acids at the N-terminus, which contribute significantly to the protein fold stability and maintenance of NP7 function. The paramagnetic form of the protein has large 1H NMR chemical shifts and broad signals due to protein aggregation. The 1H NMR and CD spectroscopy both revealed that NP7 has mainly the A heme orientation, and the mutation of Glu27 to Val27 leads to an A:B ratio change, from mainly B to mainly or almost exclusively A.NMR investigation of the ferriheme resonances of the low-spin complexes of NP2 and mutant NP2(V24E) with imidazole (ImH), histamine (Hm) and cyanide (CN-) ligands as a function of pH has been completed. Strong chemical exchange cross peaks were observed in the NOESY/EXSY spectra at low pH (pH* 5.5 to 4.0) for the three wt NP2 complexes, which indicate an interchange between two ruffling distortions of the heme. A dramatic change of A:B ratios with pH was observed for the three NP2(V24E) ligand complexes. They both are believed to be a result of a change in protein structure near E53 when it is protonated at low pH.Homo-dimers of NP1 and NP4 were investigated. 1H{15N}-HSQC NMR experimental results revealed that holo-NP4 is a homo-dimer at low pH and a mixture of dimer and monomer at high pH. In contrast, holo-NP4-Hm is monomeric at high pH (6.5 and 7.5). A H-bond between Asp30 and the Hm ligand is responsible for destabilization of the homo-dimer. NP1 was also shown to be a mixture of dimer and monomer at high pH.Heme d1 model complexes were studied. The high-spin form and low-spin forms of the Fe(III) complexes with 2-methylimidazole or imidazole, for monooxo-octaethylchlorin, trans-dioxo-octaethylisobacteriochlorin and 2,7-dioxo-octaethyliso- bacteriochlorin, were characterized by NMR spectroscopy. Spin density maps that show the orbital used for spin delocalization have been generated from Hückel calculations.Preliminary results of additional studies of the nitrophorins and future directions of study are presented.

Heme is essential for numerous enzymes involved in the regulation of vascular tone; it is an integral component of nitric oxide synthase and soluble guanylyl cyclase, and is the substrate for heme oxygenase, enzymes critical for vasodilation. Inhibition of heme synthesis is anticipated to result in a deficiency in these hemoproteins, causing disturbances in the tissue’s ability to regulate vascular tone. Hypertension is frequently associated with the morbidity of both porphyria and lead poisoning, two conditions wherein heme biosynthesis is disrupted. The hypothesis tested was, that extended pharmacological inhibition of heme synthesis disrupts normal vascular control and induces hypertension. Rats were treated with SA, a heme synthesis inhibitor, for two weeks; this depleted heme stores of the liver, kidney, spleen and vasculature by up to 62.2%. A significant decrease in hematocrit, hemoglobin, urine nitrate levels, NOS activity and sGC activity were also produced, indicating compromised hemoprotein synthesis and function. Ex vivo studies of blood vessels revealed blunted sensitivity to nitric oxide donors. Lastly, SA treatment produced a significant increase in left ventricular mass, which is indicative of altered cardiac output and blood pressure elevation. Next, telemetry devices were used to determine in vivo blood pressure and salt-sensitivity of blood pressure of rats treated with SA for 33 days. Hemodynamic changes were minimal, yet there was a mild decrease in pressure over two weeks of SA treatment alone. The change from low to high salt diet in SA rats showed no difference compared to control rats. A small increase was observed in 3 mg/kg L-NAME plus high salt compared to high salt alone, while there was no change at this dose in control animals. Heme tissue and blood content was depleted by up to 47%, but was less than two-week experiments. An increase in kidney medulla NOS activity by 19% was observed in vitro compared to controls. Initial two-week experiments were consistent with the hypothesis above, as heme depletion impaired in vivo activity of NOS and sGC and altered vasodilator function. Nevertheless, in vivo results did not support the hypothesis as hypertension and salt-sensitivity of blood pressure were not observed. / Thesis (Master, Pharmacology & Toxicology) -- Queen's University, 2008-06-19 12:32:08.686

C-type cytochromes are essential for almost all organisms and are mainly involved in electron transport; they are characterised by the covalent attachment of heme to protein through two thioether bonds to a CXXCH peptide motif. This thesis describes the development of in vitro systems to establish chemical aspects of the process of cytochrome c maturation. Initially, the uncatalysed reaction of heme and apocytochrome c from Hydrogenobacter thermophilus cytochrome c₅₅₂ was studied in vitro, yielding the desired thioether bonds under mild conditions and in the absence of any biosynthesis apparatus. The reaction proceeded via a b-type cytochrome intermediate, showing the ability of the apoprotein to bind heme noncovalently prior to thioether bond formation. It was also determined that the two cysteine residues of the CXXCH motif can form a disulfide. Optimal reaction conditions for thioether bond formation required both the heme and the cysteine residues to be reduced. Mechanistic insights were gained by showing that the thioether bonds can form independently from one another. Furthermore, it was shown that, in the case of H. thermophilus apocytochrome, thioether bonds can form stereoselectively with respect to the α, γ mesoaxis of heme. These findings were extended to a broader range of apocytochromes. It was discovered that mitochondrial apocytochromes c from horse heart and yeast also formed thioether bonds with heme, as well as the bacterial Paracoccus denitrificans apocytochrome c₅₅₀. It was established that apocytochromes show a trend to bind hydrophobic ligands or heme to yield b-type cytochromes. Thioether bonds can form in vitro in these heme-protein complexes. In the second part of this thesis the heme chaperone CcmE, which is part of the cytochrome c biogenesis apparatus in many Gram-negative bacteria, was studied as an extension to the establishment of in vitro systems. It was discovered that CcmE can bind heme initially non-covalently and then covalently upon reduction of the heme. However, CcmE seems to have a preference for ferric rather than ferrous heme. The involvement of the vinyl groups of heme is suggested. Mutation of the heme-binding histidine residue of CcmE established the involvement of this residue in the covalent binding of heme for the in vitro reaction; mechanistic insights were gained from the observation that a histidine to cysteine mutant could still bind heme covalently in vivo and in vitro. Effects of the presence of a His tag on CcmE were shown and are discussed. Furthermore, in vitro heme transfer from CcmE to certain apocytochromes c was achieved. All these in vitro results mimic, and thus have implications for, the molecular pathway of heme transfer during the complex process of c-type cytochrome maturation in vivo.

Bibliography: leaves 104-116 / iv, 116 leaves, [11] leaves of plates : ill ; 30 cm. / Title page, contents and abstract only. The complete thesis in print form is available from the University Library. / Thesis (Ph.D.)--University of Adelaide, Dept. of Biochemistry, 1987

Thesis (Ph. D.)--University of Adelaide, Dept. of Biochemistry, 1987. / Includes bibliographical references.

Thesis (Ph. D.)--University of Adelaide, 1988. / Includes bibliographical references.

Thesis (Ph. D.)--University of Adelaide, Dept. of Biochemistry, 1988.

Thesis (M. Med. Sc.)--University of Hong Kong, 2008. / Includes bibliographical references (p. 46-57)