Infrared (IR) spectroscopy is a well-established technique for probing the structure, behavior, and surroundings of molecules in their native environments. Its characteristics-most specifically high structural sensitivity, ready applicability to aqueous samples, and broad availability-make it a valuable enzymological technique, particularly for the interrogation of ion binding sites. While IR spectroscopy of the "garden variety" (steady state at room temperature with wild-type proteins) is versatile and powerful in its own right, the combination of IR spectroscopy with specialized experimental schemes for leveraging ultrafast time resolution, protein labeling, and other enhancements further extends this utility. This book chapter provides the fundamental physical background and literature context essential for harnessing IR spectroscopy in the general context of enzymology with specific focus on interrogation of ion binding. Studies of lanthanide ions binding to calmodulin are highlighted as illustrative examples of this process. Appropriate sample preparation, data collection, and spectral interpretation are discussed from a detail-oriented and practical perspective with the goal of facilitating the reader's rapid progression from reading words in a book to collecting and analyzing their own data in the lab.
