STRUCTURE SOLUTION OF AZURIN-II FROM ALCALIGENES-XYLOSOXIDANS USING THE LAUE METHOD - POSSIBILITY OF STUDYING IN-SITU REDOX CHANGES USING X-RAYS

We have recently demonstrated that X-rays can be used for changing the redox states of the metal centre in metalloproteins [Murphy et al. (1 995). J. Synchrotron Rad. 2, 64-69]. The possibility of using the Laue method for studying the structural changes associated with such X-ray -induced reactions is explored by applying the method to the structure determination of a new azurin (hereafter referred to as azurin II) fr om the denitrifying bacterium Alcaligenes xylosoxidans. Laue X-ray dif fraction data of azurin II were collected at station 9.7 of the SRS Da resbury. Three diffraction patterns were recorded on film packs at thr ee different crystal orientations. The data were processed using the D aresbury Laue Software Suite to give 2224 independent single reflectio ns (R(merge) = 0.136) in the wavelength range 0.36-1.40 Angstrom. The data completeness was 44% at 2.55 Angstrom, resolution. Phase determin ation for the data was undertaken using the molecular-replacement meth od; the top peak was chosen in both the rotation function and the subs equent translation function. This solution agreed well with the molecu lar-replacement solution achieved independently using monochromatic da ta. The electron-density map showed reasonably good agreement with the model and the copper site was readily recognizable as it had the high est density. To see if the electron-density map could be improved, the doublets in the diffraction data were then deconvoluted. This added 2 6% data in the region infinity-2d(min) resulting in an improvement in the data completeness to 50% and thus in improved continuity of the el ectron-density map. The quality of these maps is discussed from the po int of view of the suitability of this approach for studying redox-ind uced structural changes.