Electron Paramagnetic Resonance Spectroscopy

EPR spectroscopy has been an important tool in nitrogenase research for the last 50 years. The three metalloclusters in nitrogenase, the Fe protein [4Fe–4S] cluster, and the MoFe protein P-cluster, and FeMo-cofactor, all have EPR spectra when poised in the appropriate paramagnetic states. EPR spectroscopy can probe changes in the electronic properties of each metal cluster, such as when substrates bind, and can provide a definitive method for observing changes in the redox states of the clusters. In this chapter, the methods for analysis of the three metal clusters of nitrogenase by EPR spectroscopy are described, along with methods for trapping substrate-derived intermediates on the active site that are amenable to characterization by EPR and other magnetic resonance spectroscopy techniques.