Oxygen, that supports all aerobic life, is abundant in the atmosphere because of its constant regeneration by photosynthetic water oxidation, which is catalyzed by a Mn4CaO5 cluster in photosystem II (PS II), a multi subunit membrane protein complex. X-ray and other spectroscopy studies of the electronic and geometric structure of the Mn4CaO5 cluster as it advances through the intermediate states have been important for understanding the mechanism of water oxidation. The results and interpretations, especially from X-ray spectroscopy studies, regarding the geometric and electronic structure and the changes as the system proceeds through the catalytic cycle will be summarized in this review. This review will also include newer methodologies in time-resolved X-ray diffraction and spectroscopy that have become available since the commissioning of the X-ray free electron laser (XFEL) and are being applied to study the oxygen-evolving complex (OEC). The femtosecond X-ray pulses of the XFEL allows us to outrun X-ray damage at room temperature, and the time-evolution of the photo-induced reaction can be probed using a visible laser-pump followed by the X-ray-probe pulse. XFELs can be used to simultaneously determine the light-induced protein dynamics using crystallography and the local chemistry that occurs at the catalytic center using X-ray spectroscopy under functional conditions. Membrane inlet mass spectrometry has been important for providing direct information about the exchange of substrate water molecules, which has a direct bearing on the mechanism of water oxidation. Moreover, it has been indispensable for the time-resolved X-ray diffraction and spectroscopy studies and will be briefly reviewed in this chapter. Given the role of PS II in maintaining life in the biosphere and the future vision of a renewable energy economy, understanding the structure and mechanism of the photosynthetic water oxidation catalyst is an important goal for the future.

The ultra-bright femtosecond X-ray pulses provided by X-ray Free Electron Lasers (XFELs) open capabilities for studying the structure and dynamics of a wide variety of biological and inorganic systems beyond what is possible at synchrotron sources. Although the structure and chemistry at the catalytic sites have been studied intensively in both biological and inorganic systems, a full understanding of the atomic-scale chemistry requires new approaches beyond the steady state X-ray crystallography and X-ray spectroscopy at cryogenic temperatures. Following the dynamic changes in the geometric and electronic structure at ambient conditions, while overcoming X-ray damage to the redox active catalytic center, is key for deriving reaction mechanisms. Such studies become possible by using the intense and ultra-short femtosecond X-ray pulses from an XFEL, where sample is probed before it is damaged. We have developed methodology for simultaneously collecting X-ray diffraction data and X-ray emission spectra, using an energy dispersive spectrometer, at ambient conditions, and used this approach to study the room temperature structure and intermediate states of the photosynthetic water oxidizing metallo-protein, photosystem II. Moreover, we have also used this setup to simultaneously collect the X-ray emission spectra from multiple metals to follow the ultrafast dynamics of light-induced charge transfer between multiple metal sites. A Mn-Ti containing system was studied at an XFEL to demonstrate the efficacy and potential of this method.