title: Theoretische Modellierung aktiver Zentren von molybdänabhängigen Enzymen
creator: Habib, Uzma
subject: 540
subject: 540 Chemistry and allied sciences
description: Molybdenum and tungsten active site model complexes, derived from the protein X-ray crystal structure of the first W-containing nitrate reductase isolated from Pyrobaculum aerophilum, were computed for nitrate reduction at the COSMOB3LYP/SDDp//B3LYP/Lanl2DZ(p) level of density functional theory (DFT). The molybdenum containing active site model complex has a considerably larger activation energy (34.4 kcal/mol) for the oxygen atom transfer from the nitrate to the metal center as compared to the tungsten containing active site model complex (12.0 kcal/mol). Oxidation of the educt complex is close to thermoneutral (-1.9 kcal/mol) for the Mo active site model complex but strongly exothermic (-34.7 kcal/mol) for the W containing active site model complex. The low relative energy for the oxidized W metal complex makes the regeneration of the +IV oxidation state much more difficult as compared to the Mo metal complex. The MVI to MIV reduction requires much more reductive power (more negative redox potential)when the metal center M is a tungsten rather than a molybdenum atom. So, although the reduction of nitrate is stimulated when W replaces Mo in the active site of Nar the catalytic cycle breaks after the reduction of nitrate to nitrite when the biochemical reducer is not strong enough to reduce the metal center. Ethylbenzene dehydrogenase (EBDH) is an enzyme that catalyzes the oxygen-independent, stereospecific hydroxylation of ethylbenzene to (S)-1-phenylethanol. EBDH active site models, derived from protein X-ray crystal structure, were computed at the COSMOB3LYP/SDDp//B3LYP/Lanl2DZ(p) energy level of DFT in order to investigate most probable mechanism, ionic or radical pathway. In addition, different protonation states and participation of amino acid residues near to the Mo center were considered. Models with protonation of His192, Lys450, Asp223 and model without protonation were investigated for comparison. Computed relative energies indicate that the overall lowest energy barrier pathway results when ionic and radical pathways are mixed. This mechanism of ethylbenzene hydroxylation starts with a homolytic C1-Hs bond cleavage (TS1’) resulting in the formation of a radical type intermediate (I’) and then in order to continue the reaction by the easier O1Hs anion transfer, an electron needs to be transferred from the substrate to the Mo-OH moiety to transform the di-radical to the zwitter ionic intermediate. Then the transfer of O1Hs anion from the Mo to the cationic substrate (TS2) results in the formation of product bound complex (P). Among those the protonated Lys site corresponds to the energetically best pathway for the hydroxylation of ethylbenzene by EBDH. Acetylene hydratase (AH) of Pelobacter acetylenicus is a tungsten (W) containing iron-sulfur enzyme that catalyzes the transformation of acetylene to acetaldehyde. DFT studies were performed on the model complexes derived from the native protein X-ray crystal structure of AH. Based on the computational results we proposed the most likely nucleophilic mechanism for the hydration of acetylene by the acetylene hydratase (AH) enzyme. In this mechanism, the water (Wat1424) molecule is coordinated to the W center and Asp13 is assumed to be in anionic form. The Wat1424 molecule is activated by W and then donates one of its proton to the anionic Asp13 forming the W-bound hydroxide and protonated Asp13. The W-bound hydroxide then attacks the C1 atom of acetylene together with the transfer of proton from the Asp13 to its C2 atom, resulting in the formation of a vinyl alcohol intermediate complex. The energy barrier associated with this step is 14.4 kcal/mol. The final, rate limiting, step corresponds to the tautomerization of the vinyl alcohol intermediate to acetaldehyde via intermolecular assistance of two water molecules, associated with the energy barrier of 18.9 kcal/mol. An alternative, electrophilc pathway, was also considered but the energy barriers are found to be higher than for the nucleophilic pathway described here. Sulfite oxidase (SO), selenate reductase (SeR) and nitrate reductases (NRs) are among the mononuclear molybdenum enzymes involved in the catalysis of metabolic redox reactions.The active site composition of SO has one molybdopterin (MPT) ligand and it oxidizes the sulfite to sulfate, SeR has two MPT ligands and it reduces the selenate to selenite, while NRs reduces nitrate to nitrite by either one or with two MPT’s at the active site. Is the active site itself special in some way for the oxidation/reduction of one or the other substrate? Or do the different active sites behave essentially the same way and it is the role of the protein to make it specific. To clarify these, DFT studies were performed on the computational model complex, [MoVIO2(S2C2Me2)SMe]- (A, derived from the X-ray crystal structure of native SO),and on the experimental model complex [MoVIO2(mnt)2]2- (B, coordination mode similar to the active site of SeR) for the oxidation of selenite and sulfite. For the oxidation of sulfite model A which resembles the SO active site is clearly the best choice (lowest barrier, minor exothermicity). For the reduction of selenate a smaller activation is computed for model A, but the reaction is less exothermic with model B, which resembles the SeR active site. DFT computations were also carried out on simple active site model complexes of SeR to investigate different ways of binding the substrate and the OAT reaction. Unfortunately, the results are little conclusive. Larger models might be needed to obtain more meaningful computational results.
date: 2012
type: Dissertation
type: info:eu-repo/semantics/doctoralThesis
type: NonPeerReviewed
format: application/pdf
identifier: https://archiv.ub.uni-heidelberg.de/volltextserverhttps://archiv.ub.uni-heidelberg.de/volltextserver/13407/1/FinalAB.pdf
identifier: DOI:10.11588/heidok.00013407
identifier: urn:nbn:de:bsz:16-opus-134072
identifier:   Habib, Uzma  (2012) Theoretische Modellierung aktiver Zentren von molybdänabhängigen Enzymen.  [Dissertation]     
relation: https://archiv.ub.uni-heidelberg.de/volltextserver/13407/
rights: info:eu-repo/semantics/openAccess
rights: http://archiv.ub.uni-heidelberg.de/volltextserver/help/license_urhg.html
language: eng