Kinetic Modeling of the X-ray-Induced Damage to a Metalloprotein

Abstract

It is well known that biological samples undergo x-ray-induced degradation. One of the fastest occurring x-ray-induced processes involves redox modifications (reduction or oxidation) of redox-active cofactors in proteins. Here we analyze room temperature data on the photoreduction of Mn ions in the oxygen evolving complex (OEC) of photosystem II, one of the most radiation damage sensitive proteins and a key constituent of natural photosynthesis in plants, green algae and cyanobacteria. Time-resolved x-ray emission spectroscopy with wavelength-dispersive detection was used to collect data on the progression of x-ray-induced damage. A kinetic model was developed to fit experimental results, and the rate constant for the reduction of OEC MnIII/IV ions by solvated electrons was determined. From this model, the possible kinetics of x-ray-induced damage at variety of experimental conditions, such as different rates of dose deposition as well as different excitation wavelengths, can be inferred. We observed a trend of increasing dosage threshold prior to the onset of x-ray-induced damage with increasing rates of damage deposition. This trend suggests that experimentation with higher rates of dose deposition is beneficial for measurements of biological samples sensitive to radiation damage, particularly at pink beam and x-ray FEL sources.

Serial time-resolved crystallography of photosystem II using a femtosecond X-ray laser

“…13. These structural observations are consistent with spectroscopic results, which indicate that the distance between the dangler Mn and the Mn 3 O x Ca distorted cubane is indeed shorter in the dark S 1 state than in the 1.9 Å structure based on the synchrotron data, which might be influenced by X-ray induced reduction of the Mn ions in the metal cluster 18,19 . This shorter distance is in agreement with density function theory (DFT) studies 4,18,20 based on the 1.9 Å structure of PSII 6 , however, the current resolution limit of 5 Å does not allow a quantitative assessment.…”

Goniometer-based femtosecond crystallography with X-ray free electron lasers

“…This innovation provides a pathway to obtain atomic information from proteins that only form micrometer-to nanometer-sized crystals, such as many membrane proteins and large multiprotein complexes. Moreover, XFELs enable "diffraction before reduction" data collection to address another major challenge in structural enzymology by providing a means to determine catalytically accurate structures of acutely radiation-sensitive metalloenzyme active sites (6), such as high-valency reaction intermediates that may be significantly photoreduced during a single X-ray exposure at a synchrotron, even at very small doses (7)(8)(9)(10)(11). Furthermore, the use of short (tens of femtoseconds) X-ray pulses further complements the structural characterization of biochemical reaction processes by providing access to a time domain two to three orders of magnitude faster (12, 13) than currently accessible using synchrotrons.…”

Radiation chemists look at damage in redox proteins induced by X‐rays

“…Using coherent femtosecond X‐ray pulses, nine orders of magnitude more intense than synchrotron sources, three‐dimensional X‐ray crystal structures can be obtained from large numbers of microcrystals, each exposed to a single pulse. The problem of radiation damage is eliminated as data are acquired from each microcrystal before damage occurs with all but the highest intensity sources . The method is still far from routine, but it is starting to be applied to enzyme structures including metalloenzymes, and can be used at room temperature, as demonstrated by the recent determination of a structure of cytochrome c oxidase …”

“…The problem of radiation damage is eliminated as data are acquired from each microcrystal before damage occurs 79 with all but the highest intensity sources. 80 The method is still far from routine, but it is starting to be applied to enzyme structures including metalloenzymes, 81 and can be used at room temperature, as demonstrated by the recent determination of a structure of cytochrome c oxidase.…”