title: Crystallographic, spectroscopic, and computational analysis of a flavin C4a-oxygen adduct in choline oxidase.
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 A 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.
authors.affiliation: Biology Department, Brookhaven National Laboratory, Upton, New York 11973-5000, School of Chemistry and Biochemistry, Georgia Institute of Technology, Atlanta, Georgia 30332-0400, USA. amorv@bnl.gov
authors.forename: Allen M
authors.initials: AM
authors.lastname: Orville
authors.forename: George T
authors.initials: GT
authors.lastname: Lountos
authors.forename: Steffan
authors.initials: S
authors.lastname: Finnegan
authors.forename: Giovanni
authors.initials: G
authors.lastname: Gadda
authors.forename: Rajeev
authors.initials: R
authors.lastname: Prabhakar
mesh_terms: D000429:Alcohol Oxidoreductases / Q000032:analysis / Q000737:chemistry* / Q000378:metabolism; D001173:Arthrobacter / Q000201:enzymology; D001426:Bacterial Proteins / Q000032:analysis / Q000737:chemistry / Q000378:metabolism; D001665:Binding Sites / Q000502:physiology; D019295:Computational Biology / Q000379:methods; D018360:Crystallography, X-Ray / Q000379:methods; D005182:Flavin-Adenine Dinucleotide / Q000032:analysis / Q000737:chemistry* / Q000378:metabolism; D005415:Flavins / Q000032:analysis / Q000737:chemistry / Q000378:metabolism; D005420:Flavoproteins / Q000032:analysis / Q000737:chemistry* / Q000378:metabolism; D010100:Oxygen / Q000032:analysis / Q000737:chemistry* / Q000378:metabolism; D017433:Protein Structure, Secondary / Q000502:physiology; D013052:Spectrometry, X-Ray Emission / Q000379:methods
chemical_list: D001426:Bacterial Proteins; D005415:Flavins; D005420:Flavoproteins; D005182:Flavin-Adenine Dinucleotide; C008173:isoalloxazine; D000429:Alcohol Oxidoreductases; C019888:choline oxidase; D010100:Oxygen