Cluster-selective Fe labeling of a Twitch-domain-containing radical SAM enzyme

57 Fe-specific techniques such as Mössbauer spectroscopy are invaluable tools in mechanistic studies of Fe-S proteins. However, they remain underutilized for proteins that bind multiple Fe-S clusters because such proteins are typically uniformly enriched with 57 Fe. As a result, it can be unclear which spectroscopic responses derive from which cluster, and this in turn obscures the chemistry that takes place at each cluster. Herein, we report a facile method for cluster-selective 57 Fe enrichment based on exchange between the protein's Fe-S clusters and exogenous Fe ions. Through a combination of inductively coupled plasma mass spectrometric and 57 Fe Mössbauer spectroscopic analysis, we show that, of the two [Fe 4 S 4 ] clusters in BtrN (a Twitch-domain-containing radical S -adenosyl- l -methionine (SAM) enzyme), the Fe ions in the SAM-binding cluster undergo faster exchange with exogenous Fe 2+ ; the auxiliary cluster is essentially inert under the reaction conditions. Exploiting this rate difference allows for either of the two [Fe 4 S 4 ] clusters to be selectively labeled: the SAM-binding cluster can be labeled by exchanging unlabeled BtrN with 57 Fe 2+ , or the auxiliary cluster can be labeled by exchanging fully labeled BtrN with natural abundance Fe 2+ . The labeling selectivity likely originates primarily from differences in the clusters' accessibility to small molecules, with secondary contributions from the different redox properties of the clusters. This method for cluster-selective isotopic labeling could in principle be applied to any protein that binds multiple Fe-S clusters so long as the clusters undergo exchange with exogenous Fe ions at sufficiently different rates. Although the radical SAM enzyme BtrN contains two [Fe 4 S 4 ] clusters, it is shown that only one undergoes exchange with exogenous Fe ions. This enables the facile and selective 57 Fe labeling of either [Fe 4 S 4 ] cluster.