X-ray free electron lasers (XFELs) are linear accelerators that generate powerful X-rays and have proven fruitful for the structural and biochemical investigations of protein structure, function, dynamics, and kinetics in crystallo. BlaC is a beta-lactamase from Mycobacterium tuberculosis, and the first step in understanding its function was to examine its structure using an XFEL with and without ceftriaxone—a third generation cephalosporin antibiotic. Development of mix-and-inject-serial crystallography (MISC) has expanded the utility of XFELs to include enzymes, protein molecules that catalyze biological reactions; structural results indicated that this approach would be feasible with BlaC. The hydrolysis and degradation of ceftriaxone by BlaC was therefore investigated employing the MISC method. The results expose the process of antibiotic cleavage and inactivation in real time and in near atomic detail, demonstrating that this approach is both versatile and can be applied to key reactions of enzymes and other biological macromolecules. To complement these results, steady state kinetics for the BlaC reaction were also investigated by UV-visible spectroscopy, and catalytic mutants were generated. The rate parameters measured near physiological conditions agree with previously reported values, whereas those measured near the crystallization conditions are concordant with the structural observations in MISC. Additional X-ray structures were elucidated as part of this thesis. First, the cyanophage P-SSM2 encodes a viral ferredoxin (Fd) that reroutes photosynthesis in the planet’s most prevalent phototroph, Prochlorococcus marinus. This viral ferredoxin (P-SSM2 Fd) was crystallized and its structure elucidated by X-ray crystallography to 1.6 Å to demonstrate that its topology is similar to other ferredoxins based on homology alignments. Further, the electrostatic surface was compared to that of other photosynthetic Fds, which may help explain the differences in midpoint reduction potentials between them. Second, peroxins (PEX proteins) are key to the biogenesis and function of peroxisomes that sequester oxidative metabolic reactions in cells. The X-ray crystal structure of a peroxin protein complex of PEX4 and PEX22 from the reference plant, Arabidopsis thaliana, was determined. Finally, adenylate kinase serves as a model for protein dynamics, and structural investigations of this enzyme from two bacterial sources, Methanotorris igneus and Thermus thermophiles, were carried out.