X -ray absorption spectroscopy studies on redox -active manganese.

Abstract

X-ray absorption spectroscopy (XAS) has been widely used to determine solution structure of metalloproteins by probing the oxidation states and ligation of the transition metal ions. A limitation of XAS is its sensitivity to x-ray induced photoreduction. Radiation damage is a particularly severe problem for redox proteins. One solution is to make XAS measurements at low temperature. However, some samples are incompatible with cryo-protectants and radiation damage may even be accelerated by some cryo-protectants. Moreover, some proteins are known to be thermochromic (changing structure as the temperature changes), thus complete structural characterization requires variable temperature measurements, including measurements at room temperature. The focused beam and rapid scanning capabilities of the BioCAT beamline at Advanced Photon Source were used to investigate the factors that affect rates of x-ray induced photoreduction and to develop strategies that will permit the measurement of x-ray spectra for radiation sensitive samples at variable temperature. Radiation damage does not seem to show a significant dose-rate dependence. However, studies show that glycerol, a commonly used cryo-protectant, can significantly enhance photo-reduction rate. Photo-reduction is found to be reversible. A new flow XAS technique is developed and used to investigate the kinetics of an epoxidation reaction, catalyzed by a radiation sensitive Mn compound. This also permits, for the first time, variable temperature XAS measurements on biological samples under physiological conditions to elucidate the relation between the structure and reactivity of the metal sites. Flow XAS spectra of oxidized manganese superoxide dismutase (MnSOD) at room temperature were measured with no photo-reduction, allowing future study of thermochromism in MnSOD. The bound solvent of reduced native and Y34F MnSOD can be displaced by hydroxide and fluoride while the Mn site remains 5-coordinate. Utilizing with the continuous flow device, XAS spectra of intermediates of the epoxidation can be directly measured on a time frame of milliseconds. The results, based on the oxidation state of the intermediate, suggest that the active species is a Mn(IV)-O&bull; radical rather than Mn(V) = O. The pre-edge transition is the most reliable method for investigating the presence of Mn = O species, suggesting such a structure is not found in the OEC before the production of <italic>S</italic>₄.