Project Summary Metalloenzymes are an important class of proteins that constitute key components of cellular respiration, protein degradation pathways, the biosynthesis of natural products, among numerous other biological roles. A full mechanistic understanding of how metalloenzymes operate under physiologically relevant conditions is thus critical to address important health-related questions. The essential metal cofactors in these proteins constitute more than the active site, they also mediate electron transfer and provide structure. However, the many roles of metal cofactors pose a challenge to the assignment of spectral changes in operando to specific metal centers in metalloenzymes that contain multiple metal centers, especially of the same element. Spatial resolution of anomalous dispersion (SpReAD) has emerged as a powerful crystallographic probe of element- and site-specific metalloenzyme electronic structure. However, the current procedures for the measurement of SpReAD are not amenable to operando studies of metalloenzymes. SpReAD requires the measurement of several diffraction datasets across multiple wavelengths of incident light and thus has only been successfully measured at synchrotrons because they are tunable and monochromatic with energy resolution <1 eV. Yet the collection of SpReAD at synchrotrons require long data collections times at cryogenic temperatures which precludes operando interrogation of the highly reactive intermediates of metalloenzyme catalytic cycles under physiologically relevant conditions. In contrast, serial femtosecond crystallography at X-ray free electron lasers (XFELs) enables the collection of crystal structures through high-intensity, ultra-short (<35 fs) X-ray pulses that access the diffraction-before-destruction regime. Through use of sample delivery methods equipped to trigger reactions with well-defined time delays, serial femtosecond crystallography at XFELs permits measurement of time-resolved crystallographic data of metalloenzyme catalysis under physiologically relevant conditions. Collection of SpReAD at an XFEL could thus provide temporal and spatial resolution of molecular and electronic structure for complex metalloenzymes. However, unlike synchrotrons, XFELs are not monochromatic and exhibit pulse-to-pulse variability with a flux distribution over an energy range of ~30 eV. Herein, we propose to translate SpReAD from a static measurement at a synchrotron to an operando technique by embracing the native wavelength variation of XFEL pulses. We will target photosensitizer-tagged azurin as a well-defined model system for time-resolved XFEL-derived SpReAD data. Through comparisons against SpReAD data collected at a synchrotron and simultaneously measured X-ray spectra, we will validate our results. Finally, we will use this technique to identify proposed intermediates in the mechanism of nitric oxide reductase to demonstrate the broad applicability of time-resolved SpReAD to address outstandi...