Quaternary ammonium oxidative demethylation: X-ray crystallographic, resonance Raman, and UV-visible spectroscopic analysis of a Rieske-type demethylase.

Herein, the structure resulting from in situ turnover in a chemically challenging quaternary ammonium oxidative demethylation reaction was captured via crystallographic analysis and analyzed via single-crystal spectroscopy. Crystal structures were determined for the Rieske-type monooxygenase, stachydrine demethylase, in the unliganded state (at 1.6 Å resolution) and in the product complex (at 2.2 Å resolution). The ligand complex was obtained from enzyme aerobically cocrystallized with the substrate stachydrine (N,N-dimethylproline). The ligand electron density in the complex was interpreted as proline, generated within the active site at 100 K by the absorption of X-ray photon energy and two consecutive demethylation cycles. The oxidation state of the Rieske iron-sulfur cluster was characterized by UV-visible spectroscopy throughout X-ray data collection in conjunction with resonance Raman spectra collected before and after diffraction data. Shifts in the absorption band wavelength and intensity as a function of absorbed X-ray dose demonstrated that the Rieske center was reduced by solvated electrons generated by X-ray photons; the kinetics of the reduction process differed dramatically for the liganded complex compared to unliganded demethylase, which may correspond to the observed turnover in the crystal.

Citation

Kelly D Daughtry, Youli Xiao, Deborah Stoner-Ma, Eunsun Cho, Allen M Orville, Pinghua Liu, Karen N Allen. Quaternary ammonium oxidative demethylation: X-ray crystallographic, resonance Raman, and UV-visible spectroscopic analysis of a Rieske-type demethylase. Journal of the American Chemical Society. 2012 Feb 8;134(5):2823-34

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PMID: 22224443

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