Oxidation state and local structure of the photosynthetic Mn 4Ca catalytic cluster revealed via time-resolved x-ray spectroscopy
Abstract
Large-scale implementation of man-made systems based on artificial photosynthesis to harvest solar energy could lead to an abundant chemical storage of solar power in the form of hydrogen. The process of splitting water (2H2O → O2 + 4e- + 4H+) during photosynthesis requires a catalyst, the oxygen-evolving complex, or Mn4Ca cluster, located within the trans-membrane metalloprotein complex photosystem II. Characterization of sub-millisecond reactive intermediates in this system is central to understanding the catalysis involved in water splitting. X-ray absorption and emission spectroscopy techniques provide information on the atomic and electronic structure of these states respectively. Following the progression of X-ray induced damage, we demonstrate for the first time the feasibility of collecting room temperature Mn Kβ X-ray emission data on the dark stable S1 state of photosystem II in two different beam modes: ‘continuous’ monochromatic beam, and pulsed pink beam. The dosage/damage relation for ‘continuous’ beam measurements matches preliminary room temperature X-ray absorption models well. Additionally, the determined damage thresholds, likely representative of other metalloproteins, are sufficient for the analysis of electron dynamics and the catalytic mechanism. Computational modeling of protein damage kinetics in monochromatic mode are extrapolated to higher dose deposition rates. The results support the theory of ‘detection before destruction’ both for pulsed pink beam and free electron laser sources. X-ray absorption spectroscopy was also used to monitor X-ray-induced damage. Additionally, using the same sample delivery method as in the emission feasibility measurements, fine structure data were collected at room temperature for the S1 state. Data quality is much improved over previously published results and matches well with low temperature structure. Temperature dependent fluctuations in the k-space oscillation intensities, as well as R-space comparisons with simulation, do not support the current mechanistic theory of dynamic interconversion between mono and di-μ-oxo moieties. X-ray absorption fine structure simulations were created for various density functional theory models and compared with both the obtained room temperature data as well as an X-ray diffraction model simulation. An S 1 state model, most representative of the collected absorption data, was chosen. In order to learn more about the water-splitting step in particular, time-resolved Kβ emission laser pump (to advance the catalytic cycle), X-ray probe experiments were conducted in pulsed pink beam mode. For the first time ever, time-resolved X-ray emission spectra were collected on a protein. In addition, the first ever S3 → S0 transition state X-ray emission data are presented at three different time points. Room temperature data are shown for all S-states. Analysis of the lower states agrees well with limited published results. In contrast to the recent X-ray free electron laser experiments, the previously established S 1 → S2 state shift, characteristic of Mn oxidation, was clearly observed. There are no indications of additional oxidation past the S3 state, in agreement with the only other time-resolved X-ray analysis of the catalytic cycle, collected via X-ray absorption. Contrary to the lag phase reported in that previous study, as well as UV-vis experiments, we observe immediate Mn reduction, even within 50μs from the final laser flash. This likely eliminates two of the five currently supported mechanisms of water oxidation on the basis of speed, with radical coupling mechanisms or an OH- nucleophilic attack left as the strong candidates.
Degree
Ph.D.
Advisors
Pushkar, Purdue University.
Subject Area
Analytical chemistry|Nuclear physics
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