Chapter Eight - Structural Characterization of Poised States in the Oxygen Sensitive Hydrogenases and Nitrogenases

The crystallization of FeS cluster-containing proteins has been challenging due to their oxygen sensitivity, and yet these enzymes are involved in many critical catalytic reactions. The last few years have seen a wealth of innovative experiments designed to elucidate not just structural but mechanistic insights into FeS cluster enzymes. Here, we focus on the crystallization of hydrogenases, which catalyze the reversible reduction of protons to hydrogen, and nitrogenases, which reduce dinitrogen to ammonia. A specific focus is given to the different experimental parameters and strategies that are used to trap distinct enzyme states, specifically, oxidants, reductants, and gas treatments. Other themes presented here include the recent use of Cryo-EM, and how coupling various spectroscopies to crystallization is opening up new approaches for structural and mechanistic analysis.