High-Resolution X-ray Fluorescence and Excitation Spectroscopy of Metalloproteins

Abstract

A spectrograph has been developed with sufficient efficiency to make high-resolution fluorescence experiments on metalloproteins possible. The resolution of this spectrometer can reach 0.45 eV at 7.1 keV emission energy. The focus images of this multiple curved-crystal array spectrometer are presented. The chemical sensitivity of K/4 emission spectra can be used to identify chemical states, and the spin-polarized near-edge structure provides a new measure of the spin density. The high-resolution fluorescence metalloprotein studies should become routine with third-generation synchrotron facilities, and the strength of both site and spin selectivity should complement the structural information from other spectroscopies.

Observation of terahertz vibrations in Pyrococcus furiosus rubredoxin via impulsive coherent vibrational spectroscopy and nuclear resonance vibrational spectroscopy – interpretation by molecular mechanics

“…They are key electron donors in alkane oxidation pathways [2] and in superoxide reduction [3]. Apart from their biological significance, rubredoxins have often been employed as model systems for the development of spectroscopic techniques such as resonance Raman spectroscopy [4,5], EXAFS [6], L-edge spectroscopy [7], X-ray magnetic circular dichroism -XMCD [8], high-resolution X-ray fluorescence [9], resonance energy transfer -RET [10], optically detected electron paramagnetic resonance -ODEPR [11], and nuclear resonance vibrational spectroscopy -NRVS [12], that are ultimately employed on more complex metalloproteins. Rubredoxin from Pyrococcus furiosus (PfRd), the object of the current study, has an unfolding rate of $10 À6 s À1 at 100°C [13] and has served as a model for structural features underlying thermal stability in hyperthermophilic proteins.…”

High-resolution large-acceptance analyzer for x-ray fluorescence and Raman spectroscopy

“…The peak counting rate corresponds to 6 x 10 5 photons/s, which is a factor 200 more than previously reported for a Fe Kβ spectrum (The fluorescence yield of Fe is approximately 30% larger than that of Mn.) taken at similar incident flux [11]. From this comparison, it is clear that the instrument is capable to perform a qualitatively new type of spectroscopy.…”

“…6 shows the comparison of our data with electron yield data obtained at the ALS [43]. Our combined energy resolution was 2 eV FWHM, at an incident flux of 2x10 11 photons/sec. The scanning time for the spectrum was 20 sec per data point and 2 hours total (the electron yield spectrum was taken with 15 sec per point at an incident flux of 10 11 photons/sec) .…”

“…An earlier design of a high-resolution X-ray fluorescence spectrometer working at 6.5 keV is described by Stojanoff et al [10] and the first multi crystal version is described by Wang et al [11]. We have used a similar geometry, but have improved many details, most notably the solid angle, the efficiency and the scanning procedure.…”

“…Since emission and scattering is essentially isotropic, the analyzer has to combine good energy resolution with large angular acceptance. The instrument described here has evolved from previous designs [10,11] and its largely increased solid angle and efficiency makes it particularly suited for the study of very weak signals from, e.g., dilute biological samples and/or weak electromagnetic interactions.…”