Heterologous production and biophysical characterization of catabolic Nitratireductor pacificus pht-3B reductive dehalogenase

Reductive dehalogenases provide a possible route to the biotechnological remediation of widespread anthropogenic environmental organohalide contamination. These bacterial enzymes employ cobalamin and an internal electron transfer chain of two [4Fe-4S] clusters to remove halide ions from organohalides, leaving an organic molecule more amenable to further transformations. Detailed protocols for the cloning, heterologous expression, purification, crystallization and characterization of the catabolic dehalogenase from Nitratireductor pacificus pht-3B (NpRdhA) are presented, together with insight into enzyme turnover, substrate selectivity and the use of electron paramagnetic resonance (EPR) spectroscopy as an active site probe.