Metalloproteins containing manganese in a redox-active role are involved in a variety of physiologically important reactions of dioxygen metabolism. Perhaps the most complex is the Mn4CaO5 cluster that is involved in the oxidation of water to dioxygen in photosystem II (PS II), an ~500 kDa multi-subunit membrane protein complex. The water-oxidation reaction in PS II involves removal of four electrons from two water molecules, in a stepwise manner by light-induced oxidation, to produce a molecule of oxygen. PS II and the Mn4CaO5 cluster generate almost all of the dioxygen that supports aerobic life, and it is abundant in the atmosphere because of its constant regeneration by the oxidation of water. The light-induced oxidation of water to dioxygen is one of the most important chemical processes occurring on such a large scale in the biosphere. Although the structure of PS II and the chemistry at the catalytic site have been studied intensively, understanding the sequence in the chemistry at atomic-scale from light absorption to water-oxidation requires a new approach beyond the conventional steady state X-ray crystallography and X-ray spectroscopy at cryogenic temperatures. Following the dynamic changes in the structure of PS II and the Mn4CaO5 cluster at ambient conditions at physiological temperatures, while overcoming the severe X-ray damage to the redox active center is key for deriving the mechanism. The intense and ultra-short femtosecond (fs) X-ray pulses from a X-ray free electron laser (XFEL) provide an opportunity to overcome the current limitations in room temperature data collection for biological samples at traditional X-ray sources. The fs X-ray pulses allow us to acquire the signal before the sample is destroyed, thus making the light-induced snapshot study proposed here possible. The objective of this proposal is to study the protein structure and dynamics of PS II with X-ray diffraction, as well as the chemical structure and changes in the Mn4CaO5 cluster (charge and spin density, and covalency) with X-ray spectroscopy during the light-driven process of PS II. We will use the XFEL facilities at Stanford and elsewhere to collect X-ray diffraction and emission spectra simultaneously, and X-ray absorption spectra of the Mn cluster in its native and intermediates states at room temperature in a time-resolved manner, to capture short-lived intermediates and the step that includes the O-O bond formation. We have also started studying the process of assembly of the Mn cluster as repair and assembly of PS II is an essential component in nature. These studies have the potential to provide an unprecedented combination of correlated data between the PS II protein, the co-factors, and the Mn4CaO5 cluster, providing the geometric and electronic structure and the changes that occur during the catalytic cycle, all of which are necessary for a complete understanding of the mechanism of water oxidation. !
Oxygen metabolism is mediated by several metalloenzymes, and one of the most complicated and least understood of these is the oxygen-evolving complex in photosystem II containing a manganese-calcium cluster, which is the only enzyme in nature that is capable of oxidizing H2O to O2. This proposal is directed towards using the newly available ultra-fast X-ray free electron laser, to take snapshots of the geometric and electronic structural changes of photosystem II and its metal center, in real time under physiological conditions, to understand the mechanism of water oxidation and oxygen evolution.
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