An active pursuit in biochemistry is the understanding of how the structure and dynamics of protein systems regulate their biological function. In this chapter, we describe temperature-dependent hydrogen–deuterium exchange mass-spectrometry (TDHDX-MS) as a tool used to investigate the structure and dynamics of metalloenzymes in solution. While the method is applicable to many metalloproteins, this review will present TDHDX-MS studies centered on the widely represented family of mononuclear, non-heme iron containing lipoxygenase (LOX) enzymes that oxidize fatty acids to initiate biological reactions and activate cellular signaling. We will present how TDHDX has been used to describe the anisotropic nature of energy transfer in LOXs to initiate the rate-limiting hydrogen atom transfer reaction that proceeds through a tunneling process. Further, TDHDX-MS has been used detect protein motions and conformations in LOXs related to molecular recognition, protein allostery, and polymorphisms linked to disease.