3. Electronic Theses and Dissertations (ETDs) - All submissions
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Item Kinetic and thermodynamic studies of N-acetyl-cobalt(III)-microperoxidase 8: a vitamin B12a analogue(2014-02-18) Mathura, SadhnaItem A DFT study of vitamin B12 derivatives(2013-08-06) Govender, Poomani PennyDensity functional theory (DFT) and time dependent-DFT (TD-DFT) was applied to investigate the geometric and electronic properties of cobalamin (Cbl) models. Model compounds of the type, [B–(Co(III)(L)4–X)–Y]n+ were used, where B and Y were comprised of the alpha (α) and β axial ligands, (L)4 represented the equatorial ligand(s) and X was either hydrogen or a substituent of electron donating or withdrawing character, quantified by the Hammett constant (σp), at C10 of the corrin. All calculations were conducted in the gas phase or implicit solvent medium at the BP86/6-31+G(d,p) level of theory. High-resolution crystal structures of B12, extracted from the Cambridge Crystal Structural Database (CCSD), were used as the source of initial coordinates. DFT was used to explore the trans influence of the lower (α) axial ligand, the cis influence of various equatorial ligands and the cis influence of a substituted corrin ring at the C10 position on the Gibbs free energy (ΔG) and bond dissociation energies (BDEs) of the Co(III)–Cβ bond. Other geometric parameters such as ring distortion, axial bond lengths, equatorial bond lengths and partial charges on the Co metal centre, donor atom of the upper and lower axial ligands as well as the N-donor atoms of the macrocyclic ring are documented and discussed. The use of a broad range of alpha (α) ligands in the cobalamin models from charged and neutral N-donor ligands (NH3, NH2–, NH2–, NH2F, NHF–, NF2–, NH2CH3, NHCH3, NH(CH3)2, N(CH3)3), to naturally occurring amino acids or realistic models of their metal-coordinating side chains (methanethiol, dimethylsulfide, cysteine, methanethiolate, glycine, p-aminopyridine, imidazole, histidine, acetate, 2-propanol, serine and tyrosine), provided significant information on the trans influence of these ligands on the BDE of the Co(III)–C bond (upper axial ligand). The ligands NH3, NH2–, NH2–, NH2F, NHF–, NF2–, NH2CH3, NHCH3, were used to explore electronic effects while NH3, NH2CH3, NH(CH3)2, and N(CH3)3 were used to investigate steric effects. The naturally occurring amino acids or their models focused primarily on exploring why nature chooses an N-donor ligand such as histidine or imidazole instead of an S-donor or O-donor ligand that is also readily available from protein side chains. As the basicity of the α ligand increased in the series NH2F < NH3 < CH3NH2 < (CH3)2NH < (CH3)3N < NHF– < NHCH3– < NH2– < NF2– < NH2–(as assessed by the proton affinities) a normal trans influence was observed between the axial ligands. While the Co(III)–C bond was observed to increase in length, the Co(III)–Nα bond length decreased. The weakening of the Co(III)–C bond was paralleled by the decrease in the Co(III)–C BDE. On the other hand, as the steric bulk of the α ligand (NH3, NH2CH3, NH(CH3)2, and N(CH3)3) increased (assessed by the molar volume and Tolman cone angle), an inverse trans influence (in other words, simultaneous lengthening or shortening) between the upper and lower axial bonds was observed. The Co(III)–C bond showed a marginal increase in length while the Co(III)–Nα bond length steadily increased as the molar volume of the α ligand increased. Interestingly, the large difference in the Co–Nα bond length from the 5-coordinate to the 6-coordinate complex (later referred to as ΔCo–Nα(5c-6c)), paralleled the decrease of the Co(III)–C BDEs. It also became evident from calculations with the amino acids posing as α ligands that the nature of the α ligand (assessed by the absolute chemical hardness (η) of the ligand, with the greater the η value the harder the ligand) plays a major role in the labilisation of the organometallic bond. As the η of the α ligand increased, the Co(III)–C BDE increased. The trans influence of the α ligands resulted in the strengthening (hard ligand) and weakening (soft ligand) of the Co(III)–C bond, as was affirmed by the electron density at the bond critical point (bcp) of the Co(III)–C bond. The N-donor ligands (described as having an intermediate character as the η- values were between the hard and soft ligands) were found to be catalytically suitable (31.89 – 32.45 kcal mol -1), rather than the soft and hard donor ligands. The trans influence of the latter two ligands on the upper axial bond revealed a weakly and strongly bound alkyl group to the Co metal centre, giving Co(III)–C BDEs values of 29.39–32.27 kcal mol-1and 32.54–34.96 kcal mol-1, respectively. In addition to the corrin macrocycle, other equatorial ligands like cobaloxime, corrole, porphyrin, methylcobalt(III) pentaamine, [14-ane]N4, [15-ane]N4 and [16-ane]N4 were used in calculations to explore the cis influence on the labilisation of the Co(III)–C bond. These ligands included saturated and unsaturated cyclic rings. The results showed that the flexibility of the ring increased as the size of the equatorial ligand increased and thus affected the displacement of the Co(III) metal centre from the defined mean plane. This subsequently affected the strength of the organometallic bond, which paralleled the BDEs. The hydrogen atom at C10 of the corrin ring was substituted by electron donating (CH3, OH and NH2) or –withdrawing groups (NO, NO2, CN, COOH and Br) and the cis influence of these groups on the organometallic bond was investigated. A normal trans influence between the axial ligands was observed. As the electron density from the substitutents increased towards the ring, the Co(III)–C bond strengthened and the Co(III)–Nα bond weakened. The increased electron density from the C10 substituents influenced the contraction of the Co–Nα bond length. The greater difference in contraction of the Co–Nα bond length from the 5-coordinate to the 6-coordinate complex (ΔCo–Nα(5c-6c)) resulted in lower Co(III)–C BDEs. The TD-DFT method was used to generate both the absorption and circular dichroism (CD) spectra where the vertical electronic excited states of Co(III) cobalamin species that differ with respect to their upper axial ligand, including FCbl, ClCbl, BrCbl,SeCNCbl and CH3Cbl were calculated. The cis influence for each of the species was analysed within the framework of TD-DFT to assign the major spectral features, in other words, the α/β, D/E and γ bands in the predicted UV-visible spectra. These studies reveal that the “typical” and “atypical” absorption exhibit a high degree of σ-donation from the β-ligand to the Co(III) metal centre and the subsequent destabilisation of the corresponding d-orbitals of Co. Furthermore, as the donor ability of the β ligand increased, the contributions from the antibonding d z2 orbital to the HOMO increased, leading to a strong Co(III)–Nα σ-antibonding interaction, which is consistent with the observed lengthening of the same bond from FCbl, ClCbl, BrCbl, SeCNCbl to CH3Cbl.Item Molecular mechanisms of transport and metabolism of vitamin B12 in mycobacteria(2013-02-01) Moosa, AticaMycobacterium tuberculosis (MTB) encodes three enzymes that are dependent on vitamin B12–derived cofactors for activity, including a B12-dependent methionine synthase (MetH). Previously, work in the Molecular Mycobacteriology Research Unit (MMRU) demonstrated vitamin B12 auxotrophy in a mutant strain disrupted in the alternative, B12-independent methionine synthase, MetE. This observation established the ability of MTB to transport corrinoids despite the absence of an identifiable B12-specific transporter. In addition, it suggested that MTB does not synthesize vitamin B12 in vitro. Notably, bioinformatic analyses identified PPE2 as the only B12-related transport candidate in MTB, though as a putative B12-regulated cobalt transporter. PPE2 is unusual in possessing directly upstream of its predicted start codon one of only two B12-dependent riboswitches in the MTB genome, and it lies in a putative operon with B12 biosynthetic genes, cobU and cobQ1. In this study, the possibility that PPE2 functions in the transport of vitamin B12 or cobalt was investigated. Transcriptional and phenotypic data suggested that PPE2 was not involved in B12 transport. Instead, it was shown that cobalt can supplement the growth of an MTB metE mutant in liquid medium, strongly supporting the ability of MTB to synthesize B12 de novo. Moreover, the ability to utilise exogenous cobalt was dependent on functional PPE2, thereby establishing a role for a PPE-family member in cobalt assimilation in MTB. Vitamin B12 comprises a central corrin ring co-ordinated to 5,6-dimethylbenzimidazole (DMB) as α-axial ligand. Substituting DMB with adenine yields the alternate form, pseudo-B12. The ability of mycobacteria to utilize pseudo-B12 precursors (cobinamide and adenine) to support full function of B12-dependent metabolic pathways was evaluated. Although the pseudo-B12 precursors appeared to complement chemically the mycobacterial B12 auxotrophs, growth of the mutants on cobinamide alone complicated this interpretation. To address this limitation, DMB synthesis was targeted by disrupting the MTB bluB homologue, Rv0306. Neither site-directed mutagenesis of key Rv0306 residues, nor full-gene deletion was sufficient to eliminate growth on cobinamide. Instead, this observation highlights the need to establish biochemically the nature of the active B12 form synthesized and utilized by MTB under different conditions. In combination, the results presented here support the inferred flexibility of vitamin B12 biosynthesis in MTB, and reinforce the potential role of B12-dependent metabolism in mycobacterial pathogenesis.