Structural characterization of zinc sites in metalloproteins using x-ray absorption spectroscopy

In order to determine the coordinating atoms in the zinc sites of metalloproteins, a protocol has been developed which uses quantitative curve fitting analyses of the extended x-ray absorption fine structure (EXAFS) data. Theoretical phase and amplitude functions were calibrated using structurally characterized zinc models, and the resulting calibration parameters (threshold energy and scale factor) were held fixed for all subsequent fits to the data. The percent improvement in the goodness of fit as a function of the percent sulfur used in the refinement was employed to determine the number and identity of ligating atoms. In order to take into account the increase in the number of variables with inclusion of a second shell of scatterers, a 'ceiling', or upper limit for improvement due to additional variables, was calculated. This ceiling is defined as the percent improvement in the goodness of fit for refinements using two shells of sulfur scatterers relative to fits using a single shell of sulfurs. The ceiling was used to judge when the maximum in the curve was significant, warranting inclusion of a second shell of low atomic weight scatterers (oxygen or nitrogen). The curve maximizes below the ceiling when the zinc coordination sphere contains only sulfur, and above the ceiling when low atomic weight scatterers are present. When the curve maximizes above the ceiling, the percent sulfur corresponding to the curve maximum yields the number of sulfur scatterers in the zinc coordination sphere.;The zinc sites in a wide variety of metalloproteins were characterized using this protocol. The zinc binding site of MerR, a metalloprotein involved in bacterial mercury detoxification, consists of two cysteines at 2.33A and two low atomic weight scatterers at 2.08A. The zinc coordination sphere of tRNA-guanine transglycosylase and Com (an RNA binding protein) is (cys)...