The online microspectrophotometer allows direct measurement of absorption and fluorescence spectra from samples mounted on ESRF MX beamlines. It is possible to accurately position the focal spot of the microspectrophotometer to be co-incident with the path of the X-ray beam in the crystal (or solution). A number of studies employing a previous version of the instrument have been published and a very brief description of selected work is given below. Those interested are encouraged to consult the original references.








Nine consecutive data sets were collected on a single crystal revealing radiation induced elongation of a disulphide bond by 0.7 Å. Absorption spectra were measured and the appearance of a peak at 400 nm has been attributed to the formation of disulphide radicals (Weik et. al.).

Figure : Disulphide bond elongation Electron-density maps (contoured at 1.5A) and models showing X-ray induced elongation of a disulphide bond in a single crystal of TcAChE at 100K.



Phosphoserine aminotransferase



The conserved PLP-Lys Schiff base linkage was shown to be under significant chemical strain. Structural and spectroscopic analysis revealed radiation-induced deprotonation of the internal aldimine, with concomitant relaxation of the strain and distortion of the active site (Dubnovitsky et. al.).


Figure : Radiation damage of a Schiff base Absorption spectra of phosphoserine aminotransferase following increasing exposure to X-rays (A-G) concurrent with active site strain relief.











Superoxide reductase



The active centre iron of this redox enzyme was shown to be highly sensitive to X-ray induced photo-reduction. The combination of MX with online absorption measurements enabled the deign of a strategy to obtain structures of the oxidised and mixed-reduced states (Adam et. al.).


Figure : X-ray induced reduction of SOR Difference spectra of superoxide reductase following increasing X-ray irradiation during data collection (blue-red). The decay of the 650 nm peak was attributed to photo-reduction of the active centre.