XAS spectroelectrochemistry: reliable measurement of X-ray absorption spectra from redox manipulated solutions at room temperature

Abstract

The design and operation of a low-volume spectroelectrochemical cell for X-ray absorption spectroscopy (XAS) of solutions at room temperature is described. Fluorescence XAS measurements are obtained from samples contained in the void space of a 50 µL reticulated vitreous carbon (sponge) working electrode. Both rapid electrosynthesis and control of the effects of photoreduction are achieved by control over the flow properties of the solution through the working electrode, where a good balance between the rate of consumption of sample and the minimization of decomposition was obtained by pulsing the flow of the solution by 1-2 µL with duty cycle of ∼3 s while maintaining a small net flow rate (26-100 µL h(-1)). The performance of the cell in terms of control of the redox state of the sample and minimization of the effects of photoreduction was demonstrated by XAS measurements of aqueous solutions of the photosensitive Fe(III) species, [Fe(C2O4)3](3-), together with that of the electrogenerated [Fe(C2O4)3](4-) product. The current response from the cell during the collection of XAS spectra provides an independent measure of the stability of the sample of the measurement. The suitability of the approach for the study of small volumes of mM concentrations of protein samples was demonstrated by the measurement of the oxidized and electrochemically reduced forms of cytochrome c.

Redox state and photoreduction control using X-ray spectroelectrochemical techniques – advances in design and fabrication through additive engineering

“…As noted previously, despite the large area of the WE, reasonable electrochemical response can be obtained from XAS-SEC cells if due attention is given to ion migration and the geometry of the CE and RE (Best et al, 2016). Precise control over the placement of the electrodes for the 3D printed cell allows further improvement in performance.…”

“…Whilst the basic principles of the design of the XAS-SEC cell have been described previously, (Best et al, 2016) fashioned from RVC with a 2 mm diameter Ag/AgCl RE positioned with its junction close to the WE.…”

“…If the maximum safe flux is insufficient for the collection of extended XAS spectra with good signal to noise, then the flow parameters can be further refined. In contrast to measurements from a static sample, the flowing solution will have a fixed, steady state composition and will be less affected by spectral distortion resulting from time-dependent changes in sample composition induced by prolonged exposure to the beam (Best et al, 2016, Streltsov et al, 2018.…”

“…More recently we have described a small-volume flow electrochemical cell which is suitable for biological samples, e.g. cytochrome c (Best et al, 2016) and for radiation-sensitive CuAß complexes (Streltsov et al, 2018). A limitation to the more widespread use of that approach is the requirement for significant workshop facilities for the implementation of that design.…”

Choosing the optimal spectroscopic toolkit to understand protein function

“…XAS is informative for a radius of ∼5 Å from the absorbing atom and has the great advantage that a spectrum may be measured for any element for which the appropriate X-ray energy is experimentally accessible [10]. Recent work has combined electrochemistry with XAS [11] to access multiple redox states with future applicability to redox proteins. Examples of XAS to proteins include characterization of the structure and different redox states of the Mn cluster in photosystem II [12] and extensive contributions to understand the nature of the iron-molybdenum cofactor in nitrogenase (reviewed in [13]).…”