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 {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.
- Research Organization:
- BROOKHAVEN NATIONAL LABORATORY (BNL)
- Sponsoring Organization:
- USDOE SC OFFICE OF SCIENCE (SC)
- DOE Contract Number:
- AC02-98CH10886
- OSTI ID:
- 1040075
- Report Number(s):
- BNL--90057-2009-JA; KP1501010
- Journal Information:
- Biochemistry (Eaton), Journal Name: Biochemistry (Eaton) Journal Issue: 4 Vol. 48; ISSN 0006-2960; ISSN BICHAW
- Country of Publication:
- United States
- Language:
- English
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Related Subjects
60 APPLIED LIFE SCIENCES
ADDUCTS
CHOLINE
CRYOGENICS
CRYSTAL STRUCTURE
DECAY
DFT
ELECTRONIC STRUCTURE
FUNCTIONALS
Flavin C4a-adduct
HYDROGEN
ISOALLOXAZINES
KINETICS
OXIDASES
PROTONS
REACTION KINETICS
RESOLUTION
STABILIZATION
TRANSIENTS
oxygen intermediate
single-crystal spectroscopy
x-ray crystallography