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.
Flavin C4a-OO(H) and n class="Chemical">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: Michael Bannwarth; Dorothée Heckmann-Pohl; Sabine Bastian; Friedrich Giffhorn; Georg E Schulz Journal: Biochemistry Date: 2006-05-30 Impact factor: 3.162
Authors: I Schlichting; J Berendzen; K Chu; A M Stock; S A Maves; D E Benson; R M Sweet; D Ringe; G A Petsko; S G Sligar Journal: Science Date: 2000-03-03 Impact factor: 47.728
Authors: Annie Héroux; Dragana M Bozinovski; Michael P Valley; Paul F Fitzpatrick; Allen M Orville Journal: Biochemistry Date: 2009-04-21 Impact factor: 3.162
Authors: Raspudin Saleem-Batcha; Frederick Stull; Jacob N Sanders; Bradley S Moore; Bruce A Palfey; K N Houk; Robin Teufel Journal: Proc Natl Acad Sci U S A Date: 2018-04-23 Impact factor: 11.205
Authors: Allen M Orville; Richard Buono; Matt Cowan; Annie Héroux; Grace Shea-McCarthy; Dieter K Schneider; John M Skinner; Michael J Skinner; Deborah Stoner-Ma; Robert M Sweet Journal: J Synchrotron Radiat Date: 2011-03-19 Impact factor: 2.616