Spectroscopic changes observed in single crystals of choline oxidase upon x-ray irradiation at 100 K. (A) Optical spectra measured from a single crystal of choline oxidase before (blue) or after (red) an x-ray diffraction data set was collected with 180° of phi-axis rotation. The difference between the two is shown with the black line and with the scale on the right. Each spectrum was recorded with the phi axis at 0° by averaging ten spectra obtained with a 70 ms integration time and the “box-car” set to 10 pixel-CCD elements. (B) The time-dependent change of the optical absorption spectrum of another choline oxidase crystal held stationary in the x-ray beam. Spectra were recoded every ten seconds with the instrument parameters as in (A). Six reference spectra were recoded before the x-ray shutter was opened, averaged, and then subtracted from each spectrum obtained after the x-ray shutter was opened. The lower panel of the 3-D plot is a projection of the time-dependent difference spectra. (C) Difference features at 400 and 485 nm from (B) were fit to the single exponential equation: y = a*e^{(bt + c)}, where t = time in seconds and for the 400 nm data points a = -0.074012, b = 0.021617, and c = 0.070048; whereas the first two data points were excluded for the 485 nm and fit with a = 0.036755, b = 0.0087557, and c = -0.0144.

Comparison of the x-ray structures with the DFT optimized modes for the FAD C4a-OH adduct (A), or the C4a-OO(H) adduct (B). Orthogonal views of the simulated-annealing omit electron density maps (50 - 1.86 Å resolution) for the FAD isoalloxazine ring in choline oxidase (lower central portions). The mF_o - DF_c difference map (+3 σ) is shown with orange, translucent surface contours and the 2mF_o - DF_c map (1 σ) is displayed as a blue mesh. The FAD, C4a adduct, and DMSO were omitted from the model. For comparison, the refined C4a-OH and C4a-OO(H) atomic models are shown superimposed with C, N, and O atoms colored grey, blue and red, respectively. The arrows labeled X, Y and Z indicate the relative orientation of each image. An overlay of the x-ray structures with the DFT optimized modes for the FAD C4a-OH adduct and the C4a-OO(H) adduct (lower outer portions). The refined crystal structure is shown with flavin C atoms and bonds in orange superimposed with the DFT optimized C4a-OH (C atoms rendered with cyan sticks), or the C4a-OOH model (C atoms rendered with green sticks).

The proposed reaction scheme for the C4a adduct formation in single crystals of choline oxidase at 100 K. Radiolysis of solvent by x-rays yields solvated electrons and several types of reactive oxygen species (path a). An electron migrates to the high reduction potential FAD within the active site to yield the flavin semiquinone (path a). Either a hydroxyl radical (path b) or a superoxide radical (path c) then combines with the flavin semiquinone to form the C4a-OH or C4a-OO(H) species, respectively. It is also possible to form the C4a-OO(H) species via two electron reduction of the flavin, followed by reaction with O₂ (path d). Reaction of the flavin C4a-OOH species with an appropriate nucleophile (Nu:), such as dimethylsulfide, can also yield the C4a-OH adduct.