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.
Department of Physiology and Biophysics, Boston University School of Medicine, Boston, Massachusetts 02218, USA.
Kelly D
KD
Daughtry
Youli
Y
Xiao
Deborah
D
Stoner-Ma
Eunsun
E
Cho
Allen M
AM
Orville
Pinghua
P
Liu
Karen N
KN
Allen
D018360:Crystallography, X-Ray; D006899:Mixed Function Oxygenases / Q000032:analysis* / Q000378:metabolism; D008958:Models, Molecular; D010084:Oxidation-Reduction; D011392:Proline / Q000031:analogs & derivatives / Q000737:chemistry / Q000378:metabolism; D000644:Quaternary Ammonium Compounds / Q000737:chemistry* / Q000378:metabolism; D013056:Spectrophotometry, Ultraviolet; D013059:Spectrum Analysis, Raman
D000644:Quaternary Ammonium Compounds; D011392:Proline; D006899:Mixed Function Oxygenases; C003342:stachydrine