Crystallographic, Spectroscopic, and Computational Analysis of a Flavin-C4a-Oxygen Adduct in Choline Oxidase

Flavin C4a-OO(H) and C4a-OH adducts are critical intermediates proposed in many flavoenzyme reaction mechanisms, but they are rarely detected even by rapid transient kinetics methods. We observe a trapped flavin C4a-OH or C4a-OO(H) adduct by single-crystal spectroscopic methods and in the 1.86 {angs...

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Bibliographic Details
Published in:Biochemistry (Easton) 2009-02, Vol.48 (4)
Main Authors: Orville, A.M., Lountos, G. T., Finnegan, S., Gadda, G., Prabhakar, R.
Format: Article
Language:English
Subjects:
60 APPLIED LIFE SCIENCES
ADDUCTS
BASIC BIOLOGICAL SCIENCES
CHOLINE
CRYOGENICS
CRYSTAL STRUCTURE
DECAY
DFT
ELECTRONIC STRUCTURE
Flavin C4a-adduct
FUNCTIONALS
HYDROGEN
ISOALLOXAZINES
KINETICS
OXIDASES
oxygen intermediate
PROTONS
REACTION KINETICS
RESOLUTION
single-crystal spectroscopy
STABILIZATION
TRANSIENTS
x-ray crystallography
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Summary:Flavin C4a-OO(H) and C4a-OH adducts are critical intermediates proposed in many flavoenzyme reaction mechanisms, but they are rarely detected even by rapid transient kinetics methods. We observe a trapped flavin C4a-OH or C4a-OO(H) adduct by single-crystal spectroscopic methods and in the 1.86 {angstrom} resolution X-ray crystal structure of choline oxidase. The microspectrophotometry results show that the adduct forms rapidly in situ at 100 K upon exposure to X-rays. Density functional theory calculations establish the electronic structures for the flavin C4a-OH and C4a-OO(H) adducts and estimate the stabilization energy of several active site hydrogen bonds deduced from the crystal structure. We propose that the enzyme-bound FAD is reduced in the X-ray beam. The aerobic crystals then form either a C4a-OH or C4a-OO(H) adduct, but an insufficient proton inventory prevents their decay at cryogenic temperatures.
ISSN:0006-2960
1520-4995
DOI:10.1021/bi801918u