Browsing by Subject "H/D exchange"
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Item Explorations of iron-iron hydrogenase active site models by experiment and theory(2009-05-15) Tye, Jesse WayneThis dissertation describes computational and experimental studies of synthetic complexes that model the active site of the iron-iron hydrogenase [FeFe]H2ase enzyme. Simple dinuclear iron dithiolate complexes act as functional models of the ironiron hydrogenase enzyme by catalyzing isotopic exchange in D2/H2O mixtures. Density Functional Theory (DFT) calculations and new experiments have been performed that suggest reasonable mechanistic explanations for this reactivity. Evidence for the existence of an acetone derivative of the di-iron complex, as suggested by theory, is presented. Bis-phosphine substituted dinuclear iron dithiolate complexes react with the electrophilic species, H+ and Et+ (Et+ = CH3CH2 +) with differing regioselectivity; H+ reacts to form a 3c-2e? Fe-H-Fe bond, while Et+ reacts to form a new C-S bond. The instability of a bridging ethyl complex is attributed to the inability of the ethyl group, in contrast to a hydride, to form a stable 3c-2e? bond with the two iron centers. Gas-phase density functional theory calculations are used to predict the solutionphase infrared spectra for a series of CO and CN-containing dinuclear iron complexes dithiolate. It is shown that simple linear scaling of the computed C-O and C-N stretching frequencies yields accurate predictions of the experimentally determined ??(CO) and ??(CN) values. An N-heterocyclic carbene containing [FeFe]H2ase model complex, whose X-ray structure displays an apical carbene, is shown to undergo an unexpected simultaneous two-electron reduction. DFT shows, in addition to a one-electron Fe-Fe reduction, that the aryl-substituted N-heterocyclic carbene can accept a second electron more readily than the Fe-Fe manifold. The juxtaposition of these two one-electron reductions resembles the [FeFe]H2ase active site with an FeFe di-iron unit joined to the electroactive 4Fe4S cluster. Simple synthetic di-iron dithiolate complexes synthesized to date fail to reproduce the precise orientation of the diatomic ligands about the iron centers that is observed in the molecular structure of the reduced form of the enzyme active site. Herein, DFT computations are used for the rational design of synthetic complexes as accurate structural models of the reduced form of the enzyme active site.Item Investigation of the effect of intra-molecular interactions on the gas-phase conformation of peptides as probed by ion mobility-mass spectrometry, gas-phase hydrogen/deuterium exchange, and molecular mechanics(Texas A&M University, 2006-04-12) Sawyer, Holly AnnIon mobility-mass spectrometry (IM-MS), gas-phase hydrogen/deuterium (H/D) exchange ion molecule reactions and molecular modeling provide complimentary information and are used here for the characterization of peptide ion structure, including fine structure detail (i.e., cation-π interactions, β-turns, and charge solvation interactions). IM-MS experiments performed on tyrosine containing tripeptides show that the collision cross-sections of sodiated, potassiated and doubly sodiated species of gly-gly-tyr are smaller than that of the protonated species, while the cesiated and doubly cesiated species are larger. Conversely, all of the alkali-adducted species of try-gly-gly have collision cross-sections that are larger than that of the protonated species. The protonated and alkali metal ion adducted (Na+, K+ and Cs+) species of bradykinin and bradykinin fragments 1-5, 1-6, 1-7, 1-8, 2-7, 5-9 and 2-9 were also studied using IM-MS and the alkali metal ion adducts of these species were found to have cross-sections very close to those of the protonated species. Additionally, multiple peak features observed in the ATDs of protonated bradykinin fragments 1-5, 1-6 and 1-7 are conserved upon alkali metal ion adduction. It was observed from gas-phase H/D ion molecule reactions that alkali adducted species exchange slower and to a lesser extent than protonated species in the tyrosine- and arginine-containing peptides. Experimental and computational results are discussed in terms of peptide ion structure, specifically the intra-molecular interactions present how those interactions change upon alkali salt adduction, as well as with the sequence of the peptide. Additionally, IM-MS data suggests the presence of a compact conformation of bradykinin fragment 1-5 (RPPGF) when starting from organic solvent conditions. As water is added stepwise to methanolic solutions, a more extended conformation is populated. When the starting solution is composed of ≈90% water, two distinct mobility profiles are observed as well as a shoulder, indicating the presence of three gas-phase conformations for RPPGF. Gas-phase H/D exchange of [M+H]+ ions prepared from aqueous solvents show a bi-exponential decay, whereas samples prepared from organic solvents show a single exponential decay. The effect of solvent on gas-phase peptide ion structure, i.e., solution-phase memory effects, is discussed and gas-phase structures are compared to know solution-phase structures.Item Studies of the relationship of protein structure to regulation and catalysis in tyrosine hydroxylase(Texas A&M University, 2007-09-17) Sura, Giri RajuTyrosine hydroxylase (TyrH) catalyzes the rate-limiting step in the synthesis of the catecholamine neurotransmitters dopamine, epinephrine, and norepinephrine. Phosphorylation of Ser40 of rat TyrH activates the enzyme by decreasing the affinity for catecholamines. In humans, there are four different TyrH isoforms with varying lengths for the regulatory domain. DOPA and dopamine binding studies were performed on the phosphorylated and unphosphorylated human isoforms. The Kd for DOPA was increased two times upon phosphorylation of hTyrH1, but no change was seen for hTyrH4; the Kd value decreased with the increase in the size of regulatory domain. The small effect on the Kd value for DOPA upon phosphorylation of hTyrH suggests that DOPA does not regulate the activity of hTyrH. Dopamine binds very tightly and upon phosphorylation the affinity for dopamine is decreased. This Kd value decreases with the increase in the length of the regulatory domain. The crystal structures of substrate complexes of the homologous enzyme phenylalanine hydroxylase (PheH) show a large movement of a surface loop (residues 131-155) upon amino acid binding. The corresponding loop residues (175-200) in TyrH play an important role in DOPA formation. This conformational change in TyrH loop was studied with fluorescence anisotropy. Three tryptophan residues in the TyrH, at positions 166, 233, and 372, were mutated to phenylalanine, and Phe184 was mutated to tryptophan. An increase in anisotropy was observed in the presence of phenylalanine and 6-methyl-5-deazatetrahydropterin (6M5DPH4), but the magnitude of the change of anisotropy with 6M5DPH4 was greater than that with phenylalanine. Further characterization of the sole tryptophan in the loop showed a decrease in the amplitude of the local motion only in the presence of 6M5DPH4 alone. The conformational change in wild type TyrH was examined by H/D exchange LC/MS spectroscopy in the presence of the natural ligands. Time-course dependent deuterium incorporation into the loop in the presence of ligands indicated that the pterin alone can induce the conformational change in the loop irrespective of whether iron is reduced or oxidized. From these results, one can conclude that the loop undergoes a conformational change upon pterin binding, making the active site better for amino acid binding.