UC Berkeley

X-ray free-electron lasers (XFELs) enable the study of biological systems under functional conditions. This has sparked a scientific revolution, beginning around the time when the world's first hard X-ray FEL, the Linac Coherent Light Source (LCLS) at the Stanford Linear Accelerator Center (SLAC) became operational in 2009. By focusing a large number of photons into a small area, and having them all arrive within a few femtoseconds, the world of ultra-small and ultra-fast phenomena of biochemical processes opened up significantly.

These machines operate through a process called Self Amplified Spontaneous Emission (SASE), which begins from the shot noise of an electron beam. These electrons are accelerated to relativistic speeds before they are fed through a periodic arrangement of magnets, called undulators, which forces them to weave and emit bright bursts of X-ray light. The power of the light gets amplified along the length of the undulators as the electrons self-organize through the SASE process. The electrons are eventually discarded, while the ultra-short and ultra-bright pulses of X-ray light are directed towards experimental halls to serve as powerful probes in a variety of scientific investigations. Further details surrounding XFEL operation, as well as the data handling considerations which stem from high repetition rate data acquisition paired with customized detectors, are discussed thoroughly in this dissertation.

An optimal candidate for XFEL studies is Photosystem II (PS II), the enzyme responsible for catalyzing the light-induced oxidation of water to molecular oxygen. Nature has perfected this harvesting of light energy and conversion to chemical energy over billions of years. This makes it an excellent target of study that could yield insights useful for the development of clean energy. PS II contains a metal catalytic site crucial for its function, called the oxygen evolving complex (OEC; Mn4CaO5), which undergoes a variety of structural and chemical changes during its light-induced reaction cycle. The surrounding water and hydrogen-bonding network also play an important role in enzyme function via the transport of protons and substrate water. These processes can be tracked through a combination of time-resolved X-ray emission spectroscopy (XES) and serial femtosecond crystallography (SFX) at XFELs.

This dissertation covers three studies at the intersection of XFELs and PS II. The methods described may be applied to other metal containing enzymes as well, and need not be specific to PS II. Collecting time resolved SFX simultaneously with XES allows for the structural changes of the protein and the chemical state of the metal catalytic site to be bridged and reaction kinetics to be studied. Interpreting the XES data collected at XFELs from low concentration metalloenzymes can present its own challenges due to relatively low signal levels and chemical sensitivity. Therefore, this dissertation will also discuss an innovative technique at XFELs which uses the stimulated XES method. This approach, analogous to the stimulated Raman method in the optical regime, offers the potential to collect XES data with greater efficiency. It may also be used for uniquely probing the less intense, but more chemically rich, spectral transitions.

Interpreting the rich information content contained within the electron density maps collected at XFELs during SFX experiments can also bring about challenges, especially in the context of PS II's water networks; however, crystalline molecular dynamics (MD) simulations can serve as a promising computational tool to aid in the interpretation of the electron density maps. The simulation work described in this dissertation has the potential to extract more information from room temperature XFEL crystallography data, especially in the context of water dynamics and hydrogen bonding networks, and also holds the promise of improving our crystallographic refinement.