Cluster-selective 57Fe labeling of a Twitch-domain-containing radical SAM enzyme.

Abstract

57Fe-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 57Fe. 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 57Fe 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 57Fe Mössbauer spectroscopic analysis, we show that, of the two [Fe4S4] 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 Fe2+; the auxiliary cluster is essentially inert under the reaction conditions. Exploiting this rate difference allows for either of the two [Fe4S4] clusters to be selectively labeled: the SAM-binding cluster can be labeled by exchanging unlabeled BtrN with 57Fe2+, or the auxiliary cluster can be labeled by exchanging fully labeled BtrN with natural abundance Fe2+. 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.