The Oxidation of L-Tryptophan in Biology by Human Heme Dioxygenases

Tryptophan is an essential amino acid, which is catabolised via the kynurenine pathway leading to the formation of NAD. The initial and rate-limiting step of the kynurenine pathway is controlled by two hemoproteins called tryptophan 2,3-dioxygenase (TDO) and indoleamine 2,3-dioxygenase (IDO), which oxidise tryptophan to form N-formyl-kynurenine. A bacterial expression system for hTDO was characterised in Chapter 2. Kinetic, spectroscopic and redox analyses determined that significant differences exist. It was found that hTDO does not form a stable ferrous-oxy complex and that the ferric form of the enzyme was catalytically active. In addition hTDO does not discriminate against substrate binding to the ferric derivative. Site-directed mutagenesis of several active site residues and the role of each residue on substrate binding and in catalysis was examined in Chapter 3. The H76 residue was found to be involved in substrate binding. The phenylalanine variants of TDO showed that the hydrophobicity in the active site was essential for catalytic activity. Furthermore, the data showed that F72 is an important substrate binding residue. The highly conserved arginine residue was deemed to be an essential catalytic residue involved in substrate binding. The implications of these findings are discussed in terms of the current understanding of dioxygenase catalysis. In Chapter 4, it was shown that 1-methyl-tryptophan was a substrate for wild type IDO and variants of TDO and IDO, which is contrary to previous findings in dioxygenase literature. No activity was observed for wild type hTDO. Previous crystallographic studies and modelling in combination with kinetic, spectroscopic and redox analyses indicated that the distal histidine in the TDO active site causes steric clashes with 1-methyl-L-tryptophan. This information indicated exclusion of the deprotonation of the indole N1 mechanism, and an alternative reaction mechanism was presented. A new expression system for IDO with a cleavable hexahistidyl tag was constructed in Chapter 5. The analyses indicated that the recombinant protein had the same characteristics as all other mammalian IDOs and that diffraction quality crystals can be grown from the system. Crystal structures of IDO are required to further the understanding of substrate binding and the catalytic mechanism

Harmonious Properties of Uniform -Distant Trees

We prove that all uniform -distant trees are harmonious; every uniform -distant odd tree is strongly -harmonious, so is every uniform -distant even tree if the spine has even number of vertices. Also, all uniform -distant trees are sequential

Electron transfer with self-assembled copper ions at Au-deposited biomimetic films: mechanistic ‘anomalies’ disclosed by temperature- and pressure-assisted fast-scan voltammetry

It has been suggested that electron transfer (ET) processes occurring in complex environments capable of glass transitions, specifically in biomolecules, under certain conditions may experience the medium ’ s nonlinear response and nonergodic kinetic patterns. The interiors of self-assembled organic films (SAMs) deposited on solid conducting platforms (electrodes) are known to undergo glassy dynamics as well, hence they may also exhibit the abovementioned ‘ irregularities ’ . We took advantage of Cu 2+ ions as redox-active probes trapped in the Au-deposited − COOH-terminated SAMs, either L-cysteine, or 3-mercaptopropionic acid diluted by the inert 2-mercaptoethanol, to systematically study the impact of glassy dynamics on ET using the fast-scan voltammetry technique and its temperature and high-pressure extensions. We found that respective kinetic data can be rationalized within the extended Marcus theory, taking into account the frictionally controlled (adiabatic) mechanism for short-range ET, and complications due to the medium ’ s nonlinear response and broken ergodicity. This combination shows up in essential deviations from the conventional energy gap (overpotential) dependence and in essentially nonlinear temperature (Arrhenius) and high-pressure patterns, respectively. Biomimetic aspects for these systems are also discussed in the context of recently published results for interfacial ET involving self-assembled blue copper protein (azurin) placed in contact with a glassy environment