Abstract
Nitrogenase is the only known enzymatic system that converts atmospheric dinitrogen (N2) into bioavailable ammonia (NH3). The active-site cofactor responsible for this reactivity is a [(R-homocitrate)MoFe7S9C] cluster that is designated as the M-cluster. This important cofactor is assembled stepwise from a pair of [Fe4S4] clusters that become fused into a [Fe8S9C] core before additional refinements take place to complete the biosynthesis. NifB, a member of the radical S-adenosyl-l-methionine (SAM) superfamily, facilitates the conversion of the [Fe4S4] clusters (called the K-cluster) to the [Fe8S9C] core (called the L-cluster). This transformation includes a SAM-dependent carbide insertion with concomitant incorporation of an additional sulfur. While difficulties with the purification of NifB have historically prevented detailed biochemical analyses, we have developed a heterologous expression system in Escherichia coli that yields stable NifB proteins from various N2-fixing methanogenic organisms that can be used for studies. This chapter details the procedures necessary to prepare an active NifB protein. The methods used for the biochemical characterization of the SAM-dependent carbide insertion reactions are also described.
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