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.

SUBMITTER: Namkoong G 

PROVIDER: S-EPMC10337720 | biostudies-literature | 2023 Jul

REPOSITORIES: biostudies-literature