Anaerobic Infrared Spectroelectrochemical Methods for Studying Oxygen-Sensitive [FeFe] Hydrogenases

[FeFe] hydrogenases comprise an important class of H₂ evolving enzymes; however, these proteins are often oxygen sensitive and require anaerobic environments for characterization. Understanding the electrochemical relationships between various active and inactive states of these enzymes is instrumental in uncovering the reaction mechanisms of the complex six-iron active center of [FeFe] hydrogenases called H-cluster. Since states of the H-cluster exhibit distinct fingerprint-like spectra in the mid-IR range, IR spectroelectrochemical experiments provide a powerful methodological framework for this goal. This chapter describes protocols for performing Fourier-transform infrared (FTIR) spectroelectrochemical experiments on [FeFe] hydrogenases under anaerobic conditions. Topics included experimental design, data acquisition, and data analysis.