The work described in this thesis attempts to analyse the accumulation and degradation of cytochrome P-450 in Saccharomyces cerevisiae. The effect of environmental parameters such as oxygen and constituents of the growth medium have been examined here in an attempt to understand the mechanism underlying the accumulation and degradation of this enzyme. The highest level of S. cerevisiae cytochrome P-450 accumulation was recorded with a new strain NCYC 754 obtained from NCYC 240 and first investigated here. Cytochrome P-450 was found to accumulate during growth of S. cerevisiae only at high glucose concentrations under conditions of mitochondrial repression. It was found that in non-growing yeast a 100 ml 8% glucose (w/v) solution would enhance cytochrome P-450 accumulation. Scale-up of this effect in a 5 l bioreactor was attempted. In experiments on the removal of oxygen during the exponential growth of S. cerevisiae there was found to be a decline in cytochrome P-450 accumulation in which case it is suggested that oxygen may be acting as a substrate inducer of yeast cytochrome P-450. Culture shake speed was also used to control oxygen availability. An optimum shake speed was found which allowed the greatest rate of cytochrome P-450 accumulation, it was also found that the same shake speed caused the greatest rate of degradation of the enzyme during stationary phase. It was also discovered that semi-anaerobic conditions caused less degradation than aerobic conditions. The agents chloramphenicol, dinitrophenol and cycloheximide offered less protection against degradation than semi-anaerobic conditions. Ethanol was found to induce cytochrome P-450 in S. cerevisiae under conditions where cytochrome P-450 is not normally detectable. Added alkanols, other than ethanol, cause rapid degradation of cytochrome P-450 in non-growing yeast.

This thesis is largely concerned with the study of a cytochrome P-450 enzyme mimic consisting of an Iron porphyrin and iodosylbenzene. Chapter 1 consists of a review of the work that has been published on the above and other related metalloporphyrin biomimetic systems. The contents bear witness to the relatively recent interest in these particular enzyme mimics. A kinetic investigation forms the substance of the second chapter. The effect of structural and electronic changes on the iron porphyrin catalyst and the iodosylbenzene oxidant are measured in terms of their effect on the rate of cyclohexene oxidation. Other variables, such as the structure of the substrate and the nature of the solvent system, lead to a proposed mechanism. In Chapter 3 the iron porphyrin system is applied to 15,16-dihydrocyclopenta[a] phenanthren-17-one and its carcinogenic 11-methyl homologue. An attempt is made to selectively oxidise the terminal rings of the cyclopenta[a] phenanthrenes by Incorporating sterically bulky groups around the periphery of the iron porphyrin. The chemically active K-region remained the target for the majority of the iron porphyrins used, however some porphyrin substituents played an active role in encouraging the approach of the cyclopenta[a] phenanthrene substrate. Chemical modelling studies of the active site of the iron porphyrins and the question of steric hindrance as it relates to the approach of the cyclopenta[a] phenanthrenes are reported in Chapter 4.

Spectrophotometric studies have been carried out to determine the pH dependence of binding of SKF 525 A, Brietal sodium and carbon monoxide to cytochrome P-450. The optimal pH for metabolic conversion of SKF 525 A has been investigated and this agent and its major metabolite, SKF 8742 A, have been metabolised in vitro by swine and rat hepatic microsomes. A suitable gas liquid chromatography assay has been developed and used to analyse metabolic production. The effects of carbon monoxide, dithiothreitol, n-octylamine and of induction of cytochrome P-450 by phenobarbital on metabolism of SKF 525 A and SKF 8742 A have been investigated. Attempts have been made to synthesise SKF 525 AN-oxide. / KMBT_363 / Adobe Acrobat 9.54 Paper Capture Plug-in

Electron paramagnetic resonance (EPR) signals from tuna ferricytochrome c solutions were obtained between 4.2°K and 77°K, with g-values g(1) = 1.25, g(2) = 2.25, g(3) = 3.05« The g(3) line is 380 gauss wide between 4.2°K and 50°K with Gaussian shape, but has become 700 gauss wide with Lorentzian shape at 77°K. The temperature independent shape and width are best explained by a distribution of rhombic crystal field potentials (r.m.s. deviation = 11%). The Lorentzian shape arises from a short (10(-8) sec.) spin-lattice relaxation time. EPR spectra from horse heart ferricytochrome c single crystals were analysed to obtain the orientation of the g-axes relative to the crystallographic axes. The g(3)-axis was 76° from the crystal c-axis, close to the heme normal (71.5° to c-axis) determined from the 3-dimensional X-ray structure by Dickerson. The other 2 g-axes lay approximately along the N-Fe-N directions in the heme ring. An amended version of Eisenberger and Pershan's theory was used to explain the angular variation of the broad lines (300-2000 gauss) seen in the crystals—best fit was obtained with the distribution of ligand fields from the solution study plus a 1.5° variation in g-axis orientation. The undifferentiated absorption line shapes observed at 4.2°K in both solutions and single crystals were explained by the Portis theory of rapid adiabatic passage in solids. This theory was tested with a model system of charred dextrose, and found to be valid. Using the theory the relaxation time (τ) of the cytochrome c system was found to be, from the phase lag of the EPR signal relative to the magnetic field modulation, 3.8 x 10(-6) sec. at 4.2°K. τ was obtained between 4.2°K and 18°K from the rapid passage signals, and between 50°K and 70°K from the linewidth of the spectra. The temperature dependence of τ below 20°K could arise from a combination of a T(9) Raman spin-lattice relaxation process with a temperature independent spin-spin relaxation time of order 10(-8) seconds (which might arise from dipolar interactions between neighboring iron atoms). / Science, Faculty of / Physics and Astronomy, Department of / Graduate

In the first study presented here, the 2.5 Å crystal structure1 of the cytochrome <i>b₆f</i> complex obtained from the cyanobacterium Nostoc sp. PCC 7120 (pdb 4OGQ) was used as a guide for modification by site-directed mutagenesis in the cyanobacterium Synechococcus sp. PCC 7002 of the rate-limiting step in the central electron transport/proton translocation chain of oxygenic photosynthesis. This step is associated with the oxidation and deprotonation of plastoquinol on the electrochemically positive (p) side of the membrane. The mutagenesis strategy is based on structure studies of the <i>b₆f</i> complex in the absence and presence of quinol analogue inhibitors which bind and inhibit electron transport on the p-side of the thylakoid membrane. The strategy focused on two conserved prolines located on the p-side of the F-helix, proximal to the C-helix, in subunit IV of the seven subunit cytochrome <i>b₆f</i> complex. These prolines, residues 105 and 112 in the F-helix, are seen in the crystal structure to cause a bend in this helix away from the C-helix in the cytochrome b subunit. Thus, they are predicted to increase the portal aperture for the plastoquinol generated in the photosystem II reaction center complex that serves as the electron-proton donor to the [2Fe-2S] iron-sulfur protein and the pside b-heme. Changing the two prolines to alanine resulted in a decrease of 30-50 % in the logphase growth rate of the cell culture and reduction of photo-oxidized cytochrome f. The second study examines the binding thermodynamics of the cytochrome b6f complex and a purposed binding partner, PGRL1, using isothermal titration calorimetry. Proton Gradient Regulation-Like 1 (PGRL1) is thought to be necessary for efficient cyclic electron transfer, however, it’s mechanistic role is unknown. Here we examined for PGRL1 and cytochrome b6f complex binding and found there was no detectable interaction, indicating that PGRL1 is not a direct quinone/cyt b6f electron cofactor.<br>

Le cytochrome p450 CYP102A1, mieux connu sous le nom de BM3, provient de la bactérie Bacillus megaterium. Cette enzyme possède un groupement prosthétique hémique lui permettant de catalyser l’insertion d’oxygène dans un lien carbone-hydrogène menant généralement à une hydroxylation du substrat, ce qui en fait une monooxygénase. Ce genre de réaction demeure jusqu’à aujourd’hui difficile à effectuer par chimie traditionnelle ce qui confère un intérêt particulier à cette enzyme. Au contraire des autres cytochromes p450, BM3 est soluble (et non membranaire) et est naturellement fusionnée à son partenaire réductase formant ainsi une seule chaîne polypeptidique. Ainsi, au cours des dernières années, BM3 a attiré beaucoup d’attention de la part de l’industrie de la chimie fine et pharmaceutique due à son potentiel biocatalytique important. Cependant, son usage en industrie est restreint par son instabilité ainsi que par le coût prohibitif du cofacteur qui lui est nécessaire pour catalyser ses réactions, le NADPH. Cette thèse décrit le développement de différentes stratégies visant à libérer les réactions effectuées avec BM3 de leur dépendance au NADPH, tout en maximisant le rendement spécifique de la monooxygénase. En place du NADPH, deux autres cofacteurs de moindre coût furent utilisés comme alternative, soit le NADH et le N-benzyle-1,4- dihydronicotinamide (NBAH) en utilisant le mutant R966D/W1046S de BM3. Afin de maximiser le rendement spécifique de BM3, l’une des stratégies de cette thèse, l’optimisation du milieu réactionnel, repose sur deux éléments clés, soit favoriser la stabilité du cofacteur, car celui-ci est plus instable que l’enzyme elle-même, ainsi que d’abaisser au minimum la température de la réaction, car nous avons constaté que ceci avait pour effet d’augmenter le couplage entre les réactions réductase et monooxygénase et donc la stabilité de l’enzyme. L’effet net de la réaction ainsi optimisée fut d’augmenter le rendement spécifique du mutant R966D/W1046S par un facteur situé entre 2 et 2.6 en fonction du cofacteur utilisé. D’autre part, deux stratégies d’ingénierie enzymatique furent explorées afin de générer des mutations pouvant augmenter la performance de BM3. L’une d’entre elles, la mutagenèse par consensus guidé, généra une librairie de mutants de laquelle les mutants NTD5 et NTD6 furent identifiés, augmentant le rendement spécifique de l’enzyme comparativement à leur parent, R966D/W1046S, par un facteur de 5.2 et 2.3 pour le NBAH et le NADH, respectivement. L’autre stratégie explorée fut d’appliquer une pression sélective sur la bactérie Bacillus megaterium pour forcer, par évolution expérimentale, la performance de l’enzyme. De cette stratégie, un nouveau mutant de BM3 nommé DE, possédant 34 acides aminés substitués sur sa séquence, fut généré. Ce dernier a démontré une plus forte résistance aux solvants organiques ainsi qu’une augmentation de son rendement spécifique vis-à-vis le NADPH et le NADH d’un facteur de 1.23 et 1.76, comparativement à BM3 sauvage, respectivement. Les stratégies décrites dans cette thèse présentent une amélioration significative du rendement spécifique de BM3 ainsi que deux iii nouvelles méthodologies avec lesquelles une enzyme peut être optimisée et de nouvelles mutations bénéfiques identifiées. / The p450 cytochrome CYP102A1, better known as BM3, comes from the bacteria Bacillus megaterium. This enzyme possesses a prosthetic heme group enabling it to catalyze the insertion of oxygen into a carbon-hydrogen bond generally resulting in the hydroxylation of the substrate, the enzyme is therefore a monooxygenase. This type of reaction remains difficult to achieve by traditional chemistry. Unlike other p450 cytochromes, BM3 is soluble (is not membrane bound) and is naturally fused to its reductase partner forming a single polypeptide chain. As such, in recent years, BM3 has garnered much attention from the pharmaceutical and fine chemical industries, due to its high biocatalytic potential. However, its use in industry remains constrained by its instability as well as by the prohibitive cost of its cofactor, NADPH. This thesis describes the development of different strategies aiming at liberating reactions driven with BM3 from their dependence to NADPH whilst maximizing the specific yield of the monooxygenase. Instead of NADPH, two other inexpensive cofactors were used, namely NADH and N-benzyl-1,4-dihydronicotinamide (NBAH) by using the BM3 mutant R966D/W1046S. To maximize BM3 specific yield, one of the strategies used in this thesis work, the optimization of the reaction medium, rested on two key elements. Firstly, favouring the stabilization of the cofactor, as it was found to be more unstable than the enzyme itself and secondly lowering the reaction temperature as this effectively augmented oxidase/reductase reactions coupling and as such the stability of the enzyme. The net effect of the optimized reaction was to enhance the specific yield of the BM3 mutant R966D/W1046S by a factor of 2 and 2,6 depending on which cofactor was used. Two other enzymatic engineering strategies were explored to generate mutations which could enhance the performance of BM3. One of these, consensus guided mutagenesis, generated a library of mutants from which mutants NTD5 and NTD6 were identified enhancing the specific yield of the enzyme comparatively to their parent, R966D/W1046S, by a factor of 5,24 and 2,3 for NBAH and NADH respectively. The other strategy explored was to apply a selective pressure on Bacillus megaterium to force, by experimental evolution, the performance of the enzyme. From this strategy, a new mutant of BM3 called DE, possessing 34 new amino acid substitutions, was generated. This new mutant displayed a greater resistance to organic solvents as well as an augmentation of specific yields when used alongside NADPH and NADH comparatively to wild type BM3 by a factor of 1,23 and 1,76 respectively. The strategies described in this thesis allowed a significative enhancement of BM3 specific yield as well as represent two new methodologies by which new beneficial mutations can be identified.

Nitric oxide (NO) is a small, gaseous molecule that is toxic to life at high doses but serves a crucial role in biological processes at lower concentrations, including: cell signaling, immune response, and more recently, as a synthon in the biosynthesis of natural products in bacteria. Metalloenzymes are incredibly versatile catalysts that enable chemistry that often, still has no comparable laboratory reaction. TxtE, a cytochrome P450 (CYP), utilizes NO as a co-substrate along with dioxygen (O2) to catalyze the regioselective nitration of L-tryptophan (Trp) to produce 4-NO2-Trp. Work in this dissertation established that the TxtE ferric-superoxo intermediate is resistant to reduction, which facilitates its reaction with diffusible NO en route to an , as yet,-uncharacterized nitrating species. Furthermore, it is shown that an outer-sphere protein residue influences the nitration chemistry of TxtE. A Thr250Ala mutant version of TxtE characterized and found to lack all nitration ability despite maintaining cofactor incorporation and retaining competence for formation of the ferric-superoxo intermediate. Separately, experiments performed with wild-type TxtE demonstrate that analogs of Trp affect the lifetime of the ferric-superoxo intermediate and enable substrate hydroxylation. Additionally, a non-heme, diiron enzyme from Mycobacterium kansasii (MkaHLP) was previously established to possess NO peroxidase activity. In this dissertation, a Tyr54Phe mutant form of MkaHLP was characterized and found to have greatly diminished NO peroxidase activity due to the removal of the characteristic tyrosine ligand to the diiron site. Implications of this change in activity are discussed in the relevant section.

Thesis (Ph. D.)--University of Washington, 2003. / Vita. Includes bibliographical references (leaves 99-107).

Thesis (M.S.)--West Virginia University, 2004 / Title from document title page. Document formatted into pages; contains ix, 49 p. : ill. (some col.). Includes abstract. Includes bibliographical references (p. 41-48).

Thesis (Ph. D.)--West Virginia University, 2005. / Title from document title page. Document formatted into pages; contains viii, 141 p. : ill. (some col.). Includes abstract. Includes bibliographical references.