TDO utilizes b-type heme as a cofactor to activate dioxygen and insert two oxygen atoms into free L-tryptophan. We revealed two unidentified enzymatic activities of ferric TDO from Ralstonia metallidurans, which are peroxide driven oxygenation and catalase-like activity. The stoichiometric titration suggests that two moles of H2 O2 were required for the production of one mole of N-formylkynurenine. We have also observed monooxygenated- L-tryptophan. Three enzyme-based intermediates were sequentially detected in the peroxide oxidation of ferric TDO in the absence of L-Trp including compound I-type and compound ES-type Fe-oxo species. The Fe(IV) intermediates had an unusually large quadrupole splitting parameter of 1.76(2) mm/s at pH 7.4. Density functional theory calculations suggest that it results from the hydrogen bonding to the oxo group. We have also demonstrated that the oxidized TDO was activated via a homolytic cleavage of the O-O bond of ferric hydroperoxide intermediate via a substrate dependent process to generate a ferrous TDO. We proposed a peroxide activation mechanism of the oxidized TDO. The TDO has a relatively high redox potential, the protonated state of the proximal histidine upon substrate binding as well as a common feature of the formation of ferric hydroxide species upon substrate or substrate analogues binding. Putting these together, we have proposed a substratebased activation mechanism of the oxidized TDO. Our work also probed the role of histidine 72 as an acid-base catalyst in the active site. In H72S and H72N mutants, one water molecule plays a similar role as that of His72 in wild type TDO.
MauG is a c-type di-heme enzyme which catalyze the biosynthesis of the protein-derived cofactor tryptophan tryptophylquinone. Its natural substrate is a monohydroxylated tryptophan residue present in a 119-kDa precursor protein of methylamine dehydrogenase (MADH). We have trapped a novel bis-Fe(IV) intermediate from MauG, which is remarkably stable. A tryptophanyl radical intermediate of MADH has been trapped after the reaction of the substrate with the bis-Fe(IV) intermediate. Analysis by high-resolution size-exclusion chromatography shows that MauG can tightly bind to the biosynthetic precursor and form a stable complex, but the mature protein substrate does not.
INDEX WORDS: Tryptophan 2,3-dioxygenase, MauG, Hydrogen peroxide, Ferryl species, Bis-Fe(IV) intermediate, Ferric hydroperoxide, Ferric hydroxide, Protein radical, Acid-base catalyst, Protein-substrate complex, Mössbauer spectroscopy, Electron paramagnetic resonance sepectroscopy, Site-directed mutagenesis
|School:||Georgia State University|
|School Location:||United States -- Georgia|
|Source:||DAI-B 71/05, Dissertation Abstracts International|
|Keywords:||Ferryl species, Hydrogen peroxide, Oxygenation, Tryptophan|
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