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

Journal Article · Tue Feb 03 00:00:00 EST 2009 · Biochemistry (Eaton)

DOI:https://doi.org/10.1021/bi801918u· OSTI ID:1040075

Orville, A M; Lountos, G T; Finnegan, S; Gadda, G; Prabhakar, R

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 Lab. (BNL), Upton, NY (United States)
• Sponsoring Organization: USDOE SC OFFICE OF SCIENCE (SC)
• DOE Contract Number: DE-AC02-98CH10886
• OSTI ID: 1040075
• Report Number(s): BNL-90057-2009-JA; BICHAW; R&D Project: BO-070; KP1501010; TRN: US201210%%262
• Journal Information: Biochemistry (Eaton), Vol. 48, Issue 4; ISSN 0006-2960
• Country of Publication: United States
• Language: English

Related Subjects

59 BASIC BIOLOGICAL SCIENCES
60 APPLIED LIFE SCIENCES
ADDUCTS
CHOLINE
CRYOGENICS
CRYSTAL STRUCTURE
DECAY
ELECTRONIC STRUCTURE
FUNCTIONALS
HYDROGEN
ISOALLOXAZINES
KINETICS
OXIDASES
PROTONS
REACTION KINETICS
RESOLUTION
STABILIZATION
TRANSIENTS
Flavin C4a-adduct
DFT
oxygen intermediate
x-ray crystallography
single-crystal spectroscopy