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This thesis reports the characterization of two members of a superfamily of enzymes, namely CYP51 from the human pathogen Mycobacterium tuberculosis and CYP116B1 from the soil bacterium Ralstonia metallidurans . CYP51 is one of twenty P450s encoded in the M. tuberculosis genome and has been demonstrated to demethylate sterol compounds. In this study, the spectroscopic and biophysical properties of CYP51 are extensively characterized, focussing on the unusual collapse of its carbon monoxy-ferrous form to a novel, protonated species, and interactions with a proposed physiological redox partner: a ferredoxin protein found adjacent to CYP51 in the M. tuberculosis genome. Additionally, the plausibility of this enzyme as a target for novel anti-tubercular agents, particularly azole-antifungal derivatives, is assessed. The study of CYP116B1 describes initial characterization of a novel fusion of a cytochrome P450 to its flavin mononucleotide- and iron sulphur cluster-containing redox partner protein, which is of potential biotechnological interest due to its proposed role in thiocarbamate herbicide degradation. Assessment of the spectroscopic properties of this enzyme are reported, including electron paramagnetic resonance and resonance Raman investigations of the heme macrocycle, in addition to kinetic studies of the electron transfer capabilities of the reductase component. Furthermore, the search for a substrate for this P450 and subsequent LCMS product analysis assays with the thiocarbamates EPTC and vernolate are detailed. The dissection of CYP116B1 into its constituent domains, in order to facilitate studies of the P450 and reductase components in isolation and further resolve structural, spectroscopic, and thermodynamic properties of this enzyme is also reported.

TTQ-dependent aromatic amine dehydrogenase (AADH) from Alcaligenes faecalis has been expressed heterologously in Paracoccus denitrificans via the cloning of the aromatic amine dehydrogenase utilizing genes (aau) under the regulation of the mauF promoter, another TTQ-dependent methylamine dehydrogenase (MADH). The recombinant AADH obtained during this study is completely active indicating complete biosynthesis of the TTQ cofactor by restricted cloning of only five genes (aauBEDA and orf-2) of the nine known genes which encode AADH. Chemical and physical characterization as well as kinetic analysis, show that the recombinant AADH expressed in Paracoccus denitrificans is indistinguishable from the native AADH expressed in Alcaligenes faecalis. The catalytic small subunit of AADH has been isolated and studied in the absence of the large subunit. Separation from the holoenzyme was achieved by chemical denaturation. The separation small subunit was refolded, characterized and studied using a number of different kinetic approaches. The small subunit shows a fully integrated and completely redox active TTQ centre. The ability of the separated small subunit to oxidase amine substrate was demonstrated, despite the very slow reaction rates when compared to the holoenzyme. The altered catalytic potency of the TTQ-containing small subunit revealed the significant role of the large subunit in providing the enzyme's catalytic power. Structural X-ray crystallographic studies were performed on crystalline enzyme in an attempt to elucidate further details about the reaction mechanism of enzyme and specifically the mode of C-H bond breakage. Structures of AADH in complex with different para substituted benzylamines were obtained. These structures provide valuable datasets for future studies using computational methods to address specific aspects of the chemistry of TTQ reduction by amine substrates.

Cytochrome P450 BioI is isolated from the soil bacterium Bacillus subtilis. It is located in an operon that encodes genes involved in the synthesis of the vitamin biotin. From knock-out studies it was concluded that BioI catalyses an early step in the biosynthetic pathway, forming a precursor to the isolatable intermediate pimelic acid. This thesis covers the characterisation of this P450 and its various endogenous redox partners. The data presented here reports upon the bio-physical properties of the P450 including information on the redox potentials, how its environment affects its properties and the factors that govern substrate binding. To produce a catalytically active BioI redox system, two flavodoxins were cloned from B. subtilis and their over-expression and preparation is described. These flavodoxins have been characterised according to their redox and structural properties. The ability of the two flavodoxins to reconstitute the oxidase activity of BioI was examined by kinetic and spectroscopic techniques culminating in the analysis of the monooxygenation of various fatty acids. To complete the redox chain two NADPH-oxidoreductase candidates and a di-flavin reductase from B. subtilis were assessed and their ability to complement the P450 BioI-flavodoxin system examined.

This work presents the crystal structure determination of the transcriptional regulator CprK and of individual domains of the multidomain cytochrome P450-BM3. The crystal structure of the A264E mutant heme domain was determined with and without substrate present. Surprisingly, the structures reveal the protein to exhibit a substrate bound conformation regardless of the presence of substrate. This has provided further evidence that substrate binding leads to a dramatic shift in the equilibrium of conformational states available to the protein. In addition, the crystal structure of the C773A mutant flavin binding domain has been determined both in presence and absence of NADP+. Together with the already available structures of the other domains, this now allows both modelling and further solution studies of the full length cytochrome P450-BM3 structure.;Dehalogenans sp. are capable of using a range of chlorophenolic compounds as terminal electron acceptors in a respiratory metabolism known as halorespiration. This process is under transcriptional control by CprK, a member of the CRP-FNR family of transcriptional regulators. The crystal structure of D. hafniense CprK in complex with o-chlorophenolacetic acid (OCPA) reveals tightly bound effector molecules. Binding of OCPA is analysed through both mutagenesis and fluorescence quenching binding studies. The results have led to the hypothesis that CprK uses the bound phenolic compound pKa as an additional mechanism to sense the presence of the chloride atom. The structure presents a structural framework for further studies of the mechanism of this family of transcriptional regulators and of CprK homologues in particular.

The cloning of two open reading frames encoding a heterodimeric protein related in sequence to bacterial sarcosine oxidase and dimethylglycine oxidase from hyperthermophilic Pyrococcus furiosus DSM 3638 is described. the protein was overexpressed in E. coli, purified to homogeneity and identified as a flavoprotein by virtue of the enzymes UV-visible absorption spectrum showing characteristic lambdamax at 367 and 450 nm. The physiological substrate was identified as L-proline (t ½ = ∼105 s-1 for bleaching of flavoprotein spectrum at 450 nm). Additionally, the enzyme oxidises L-pipecolic acid ( t½ = ∼110.5 s-1) and, to a lesser extent sarcosine (t½ = ∼654 s-1). No reactivity with sodium sulfite was detected, consistent with the enzyme belonging to the flavoprotein dehydrogenase class. These data classified the enzyme as a novel hyperthermophilic L-proline dehydrogenase.;The crystal structure of PRODH at 3.3 A resolution shows the enzyme is a heterooctamer (alphabeta)4. The holoenzyme contains one mol each of FAD, FMN and ATP per mol of alphabeta complex. Isolation of monoflavinylated enzyme containing a single FAD cofactor permitted detailed redox potentiometry and pH-dependence studies of the reaction with L-proline. A bell-shaped dependence for kcat/K m as a function of pH was observed with macroscopic pK a values (7.0 +/- 0.2 and 7.6 +/- 0.2) attributed to residue ionisations in the free enzyme. The pH dependence of k cat is sigmoidal, with maximum activity realised in the alkaline region; the dependence is described by a macroscopic pK a value of 7.7 +/- 0.1 and by analogy with other flavoenzymes is tentatively attributed to the ionisation of L-proline in the Michaelis complex. Studies with H225A, H225Q and Y251F mutants ruled out the participation of these residues as catalytic bases.

The thesis describes mechanistic studies of the bacterial protein morphinone reductase (MR) using mutagenesis, kinetic and spectroscopic methods. The roles of seven active site residues were investigated. Cys-191 was initially predicted to be an active site acid; however, mutagenesis studies revealed that Cys-191 does not act in such role. His-186 and Asn-189 were shown to be involved in ligand binding. Mutation of His-186 to alanine has dramatic effects on the enzyme in the steady-state and in the oxidative half-reaction suggesting that His-186 could be an active site acid; however, pH dependence and NMR spectroscopic studies proved that His-186 could not play such role. No charge-transfer complex was observed between the H186A mutant and b-NADH in the reductive half-reaction. Mutation of Asn-189 affects enzyme kinetics and results in a slower rate of flavin oxidation. Faster rates for flavin reduction in the N189A mutant suggest that mutation of Asn-189 favours orientation of the flavin and b-NADH for hydride transfer. Neither Tyr-72 nor Tyr-356 is an active site acid. Mutation of Tyr-72 does not substantially affect enzyme kinetics. Mutations of Tyr-356 and Trp-106 had pronounced affects on enzyme kinetics, suggesting that each mutation alters the active site geometry. In the seven MR mutants studied, the flavin redox potentials are not altered compared to wild-type enzyme except for the T32A mutant where a 50 mV decrease is observed. Mutation of Thr-32 produced enhanced activity in the oxidative half-reaction and a slower activity in the reductive half-reaction effect of copper on MR was investigated. His-186 is shown to be involved in copper binding. 1-Nitrocyclohexene is a potential substrate for MR and the underlying mechanism requires further study.

Cytochrome oxidase is the terminal proton pumping enzyme of the respiratory chain, catalysing the reduction of oxygen to water. This complex enzyme is composed of up to thirteen subunits of both nuclear and mitochondrial genetic origin, but the order in which they assemble is not fully understood. To investigate assembly I utilised blue native gel electrophoresis to analyse Saccharomyces cerevisiae strains with mutations in cytochrome oxidase causing assembly defect. I identified novel subcomplexes including, for the first time, a subcomplex containing mitochondrially encoded subunits. The respiratory chain is a target of the free radical nitric oxide (NO), which reversibly inhibits cytochrome oxidase through competition with oxygen at its active site. NO also has a myriad of other targets, and many of its actions are mediated through reactive nitrogen species (RNS) formed on reaction of NO with other species. NO is released as part of the immune response to infection and has antimicrobial action. Here, I found that prolonged incubation of yeast with an NO donor inhibited growth and caused a decrease in cytochrome oxidase content in dividing cells, which may be due to NO interfering with the assembly of the enzyme. I have also analysed gene expression in yeast after short NO exposure, to gain insight into the stress sensed by the cell and the transcription factors involved. The data suggest that NO causes a general stress response, in addition to specific effects such as repression of respiratory chain genes and activation of antioxidant/detoxification systems. Many genes known to be regulated by Haplp were repressed, suggesting that NO might interfere with this transcription factor.

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