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

Journal Article · Thu Jan 01 00:00:00 EST 2009 · Biochemistry

DOI:https://doi.org/10.1021/bi801918u· OSTI ID:980126

Orville, A; Lountos, G; 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). National Synchrotron Light Source
• Sponsoring Organization: Doe - Office Of Science
• DOE Contract Number: DE-AC02-98CH10886
• OSTI ID: 980126
• Report Number(s): BNL-93044-2010-JA; TRN: US201015%%1511
• Journal Information: Biochemistry, Vol. 48, Issue 4
• Country of Publication: United States
• Language: English

Related Subjects

08 HYDROGEN
36 MATERIALS SCIENCE
37 INORGANIC
ORGANIC
PHYSICAL AND ANALYTICAL CHEMISTRY
ADDUCTS
CHOLINE
CRYOGENICS
CRYSTAL STRUCTURE
CRYSTALS
DECAY
DENSITY
ELECTRONIC STRUCTURE
ENERGY
FUNCTIONALS
HYDROGEN
ISOALLOXAZINES
KINETICS
OXIDASES
PROTONS
REACTION KINETICS
RESOLUTION
STABILIZATION
TRANSIENTS
national synchrotron light source