X-ray absorption spectroscopy of biological photolysis products: kilohertz photolysis and soft X-ray applications

In this study, we report the first kilohertz laser photolysis of carbonmonoxy myoglobin (MbCO), studied by hard X-ray absorption spectroscopy (XAS, iron K-edge at ∼7 keV). In addition, traditional frozen photolysis of MbCO has also been evaluated, alternatively, with soft X-ray absorption spectroscopy (iron L-edge at <1 keV). Changes in the K absorption edge position, the pre-K 1s-3d feature and the K-edge XANES spectra have been clearly observed following the photodissociation of MbCO, which shows the structural/electronic changes in between MbCO and its photolysis product Mb*CO, and demonstrates the advanced capacities of our apparatus in monitoring and/or characterizing biological photolysis products. On the other hand, the iron L-edge XAS have shown clearer differences (than K-edge XAS) on the electronic information between the bound MbCO and the photolyzed Mb*CO. The results from these two approaches have been compared and discussed. Practical issues related to the use of kilohertz laser with biological samples, and with synchrotron radiation beamlines, have also been discussed.