Crystallographic, spectroscopic, and computational analysis of a flavin C4a-oxygen adduct in choline oxidase

Allen M. Orville, George T. Lountos, Steffan Finnegan, Giovanni Gadda, Rajeev Prabhakar

Research output: Contribution to journalArticle

48 Citations (Scopus)

Abstract

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 Å 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.

Original languageEnglish
Pages (from-to)720-728
Number of pages9
JournalBiochemistry
Volume48
Issue number4
DOIs
StatePublished - Feb 3 2009

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choline oxidase
X-Rays
Oxygen
X rays
Crystal structure
Microspectrophotometry
Flavin-Adenine Dinucleotide
Cryogenics
Electronic structure
Density functional theory
Protons
Hydrogen
Catalytic Domain
Hydrogen bonds
Stabilization
Single crystals
Equipment and Supplies
Crystals
Kinetics
Temperature

ASJC Scopus subject areas

  • Biochemistry

Cite this

Crystallographic, spectroscopic, and computational analysis of a flavin C4a-oxygen adduct in choline oxidase. / Orville, Allen M.; Lountos, George T.; Finnegan, Steffan; Gadda, Giovanni; Prabhakar, Rajeev.

In: Biochemistry, Vol. 48, No. 4, 03.02.2009, p. 720-728.

Research output: Contribution to journalArticle

Orville, Allen M. ; Lountos, George T. ; Finnegan, Steffan ; Gadda, Giovanni ; Prabhakar, Rajeev. / Crystallographic, spectroscopic, and computational analysis of a flavin C4a-oxygen adduct in choline oxidase. In: Biochemistry. 2009 ; Vol. 48, No. 4. pp. 720-728.
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